Nucleic acid molecules encoding phytase and pH2.5 acid phosphatase

ABSTRACT

The present invention provides a nucleic acid molecule encoding a phytase. The present invention also provides a nucleic acid molecule encoding a pH 2.5 acid phosphatase. Also provided are vectors, host cells, and a method of overexpressing phytate degrading enzymes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 07/923,724, filedJul. 31, 1992, which is a CIP of Ser. No. 07/496,155, filed Mar. 19,1990, now U.S. Pat. No. 5,273,887, which is a continuation of Ser. No.07/044,077, filed Apr. 29, 1987 (abandoned).

BACKGROUND OF THE INVENTION

The mesophilic filamentous fungus Trichoderma reesei is very efficientin secreting cellulase enzymes into the growth medium. In optimizedcultivation conditions amounts, up to 40 g/l of extracellular cellulasehave been reported (Durand et al., Enzyme Microb. Technol. 10:341-346(1988); Durand et al., in Biochemistry and Genetics of CelluloseDegradation, Academic Press, 1988, pp. 135-151).

Development of transformation systems for T. reesei (Knowles et al.,EP244,234; Penttila et al., Gene 61:155-164 (1987); Berka et al.,EP215,594) has made possible the application of genetic engineeringmethods to the fungus. By genetic engineering, production profiles ofdifferent cellulase enzymes have been modulated e.g., to give strainswith improved levels of the endoglucanase I enzyme. The strong cbh1promoter has been applied to promote endoglucanase expression(Nevalainen et al., "The molecular biology of Trichoderma and itsapplication to the expression of both homologous and heterologousgenes," in Molecular Industrial Mycology, Leong and Berka, eds., MarcelDekker Inc., New York, pp. 129-148 (1991); and Harkki, A. et al., EnzymeMicrob. Technol. 13:227-233 (1991)).

In addition to tailoring the production profiles of homologous proteins,the production potential of T. reesei has been harnessed to expressvarious heterologous proteins in the fungus. So far examples are few andinclude e.g., calf chymosin (Knowles et al., EP244,234; Berka et al.,EP215,594; Harkki, A. et al., Bio/Technol. 7:596-603 (1989); Uusitalo,J. M. et al., J. Biotechnol. 17:35-50 (1991)), CBH1-Fab fusionantibodies raised against 2-phenyl-oxazolone (Nyyssonen et al.,WO92/01797) and a fungal ligninolytic enzyme (Saloheimo, M. andNiku-Paavola, M.-L. Bio/Technol. 9:987-990 (1991)). For improvedexpression the desired gene has been inserted into a cbh1 expressioncassette and introduced into T. reesei by protoplast transformation(Harkki, A. et al., Bio/Technol. 7:596-603 (1989); Nyyssonen et al.,WO92/01797; (Saloheimo, M. and Niku-Paavola, M.-L. Bio/Technol.9:987-990 (1991)). Even though heterologous filamentous fungal promoterssuch as Aspergillus amdS, argB and glucoamylase (GA) can function in T.reesei at least to some extent (Penttila et al., Gene 61:155-164 (1987);Knowles et al., EP244,234) efficient expression requires the use of ahomologous promoter. In addition, better yields have been obtained insome cases by producing the desired gene product as a fusion protein(Harkki, A. et al., Bio/Technol. 7:596-603 (1989); Nyyssonen et al.,WO92/01797). The yields of heterologous proteins obtained from T. reeseihave varied between 10-150 mg/l.

Phytate, a storage form of phosphorus in plant seeds, is part of humanand animal diets. Phytate phosphorus is poorly available tomonogastrics, because it forms complexes with multivalent metal ions andbinds to proteins. Thus degradation of phytate is of interest. Plantphytin degrading enzymes phytase and acid phosphatase for the conversionof phytate to inositol and inorganic phosphorus are produced e.g., bybacteria (Powar, V. K. and Jagannathan, V. J., J. Bacteriol.15:1102-1108 (1982); Cosgrove, D. J., Aust. J. Biol. Sci. 23:1207-1220(1970) and Cosgrove, D. J. et al., Aust. J. Biol. Sci. 23:339-343(1970); yeasts (Nayini, N. R. and Markakis, P., LebensmittelWissenschaft und Technologie. 17:24-26 (1984)) and filamentous fungicomprising several Aspergillus species such as A. terreus (Yamada etal., Agric. Biol. Chem. 32:1275-1282 (1968), A. ficuum (Gibson, D. M.Biotechnol. Lett. 9:305-310 (1987) and A. niger (Shieh, T. R. and Ware,J. H., Appl. Microbiol. 16:1348-1351 (1968)). For complete degradationof plant phytin, both phytase and pH 2.5 acid phosphatase are needed.

Industrial applications involve remarkable higher production yields thanthe amounts produced by the natural reported strains. The gene codingfor phytase has been recently isolated and characterized from A. ficuum(Van Gorcom et al., EP420,358 or WO91/05053) and the production ofphytase has been improved in A. ficuum by multiplying the copy number ofthe gene in an expression cassette containing a strong homologousAspergillus promoter e.g., GA (Van Gorcom et al., EP420,358 orWO91/05053). A gene coding for acid phosphatase has been isolated andcharacterized from A. niger, (MacRae et al., Gene 71:339-348 (1988)).

SUMMARY OF THE INVENTION

Recognizing the need for better production methods of phytase and pH2.5acid phosphatase, and for compositions containing the same, theinventors have developed highly efficient methods for the recombinantproduction thereof.

According to the invention, there is first provided a method foroverexpressing phytate degrading enzymes in Trichoderma.

There are further provided methods for overexpressing recombinantAspergillus niger phytase and pH2.5 acid phosphatase enzymes inTrichoderma and secreting such enzymes therefrom.

There are further provided expression vectors containing geneticsequences encoding such enzymes, and Trichoderma host cells transformedwith such expression vectors.

There are further provided compositions comprising one or more of theTrichoderma-synthesized, recombinant phytase-degrading enzymes of theinvention.

There are further provided methods for the use of such compositions infeed and other such methods comprising food compositions, especially foranimals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Sequence of peptide #816 ( SEQ ID No. :57:!), oligo PHY-31 ( SEQID No. :64:!), peptide #1110 ( SEQ ID No. :62:!), oligo PHY-34 ( SEQ IDNo. :65:!) and oligo PHY-35 ( SEQ ID No. :52:!). pH2.5 acid phosphataseoligonucleotide PHY-31 is a 17mer mixture with 64 fold degeneracy and asingle inosine. Peptide #816 is derived from an endoproteinase Lys-Cdigestion of purified native acid phosphatase. PHY-34 is a 17mer mixturewith 128 fold degeneracy. PHY-35 is a 17mer mixture with 64 folddegeneracy. Both PHY-34 and PHY-35 are necessary for completerepresentation of Peptide #1110. Peptide #1110 is derived from a trypsindigestion of purified native acid phosphatase.

FIGS. 2(A-C). Nucleotide sequence from the 2.1 kb SphI fragmentcontaining the pH 2.5 acid phosphatase gene SEQ ID No. :1:! with deducedamino acid translation SEQ ID No. :2:!. The intron donor, lariat andacceptor sequence as determined by cDNA sequencing are overlined. Thenucleotide sequence corresponding to peptides #816 ( SEQ ID No. :57:!)and #1110 ( SEQ ID No. :62:!) is underlined. The genomic nucleotidesequence was determined by the M13-dideoxy method (Sanger, F., et al.,Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977) with the use of theUnited States Biochemical Sequencase II kit.

FIG. 3. The amino acid sequences of the phytase tryptic peptides #792SEQ ID No. :43:! and #420 SEQ ID No. :23:! and the deducedoligonucleotides SEQ ID Nos. :3:, :4:, :5: and :6:! used in theproduction of the phytase probe by nested PCR amplification.

FIG. 4. Plasmid pALK169. The map of pALK169 containing the 2.4 kb SphIinsert and showing the restriction map of the insert. The location ofthe phytase gene is shown by an arrow. The hybridization site for the350 bp PCR fragment in the phytase sequence is shown by an intensifiedline.

FIGS. 5(A-D). The nucleotide sequence of the phytase gene. SEQ ID Nos.:7: (DNA) and :8: (amino acid)!.

FIG. 6. The PCR primers used for making the cbh1-phytase fusionfragments SEQ ID Nos. :9: and :10: and :11: and :12:!.

FIGS. 7(A-B). Construction of pALK171. The phytase gene with its ownsignal sequence was fused to the cbh1 promoter. Only the relevantrestriction sites are shown.

FIGS. 8(A-B). Construction of pALK172. The phytase gene was fused to thecbh1 signal sequence. Only the relevant restriction sites are shown.

FIG. 9. Plasmids pALK173A and pALK173B. The maps of the plasmidscontaining the phytase gene with its own promoter and the selectionmarker, amdS gene, are shown. In the plasmid, pALK173A thetranscriptional orientation of the phytase and amdS genes is the same;and in the plasmid pALK173B, the transcriptional orientation of thesetwo genes is opposite to each other.

FIG. 10. Western blots of the samples from the culture supernatants ofthe Trichoderma host strains and transformants producing phytase. Lane1: 50 ng of purified Aspergillus ALKO 243 phytase; Lane 2: 15 ng ofendoF-treated Aspergillus ALKO 243 phytase; Lanes 3 and 10: T. reeseiALKO 233; Lanes 4-5 and 11-12: T. reesei ALKO 233 transformant 171FR/A4and A13, respectively; Lanes 6 and 13: T. reesei ALKO 2221; Lanes 7-8and 14-15: T. reesei ALKO 2221 transformant 171FR/A5 and A9,respectively; Lane 9: T. reesei ALKO 2221 transformant D2; Lane 16: T.reesei ATCC56765; Lanes 17, 18, 19: T. reesei ATCC56765 transformants171FR/A21, A11, and A23, respectively. In each case 2 μl of 1:10dilution of the culture supernatant were run in the gel. 171FR: the hosttransformed with the XbaI fragment from the plasmid pALK171.

FIG. 11. The PCR primers used for making the cbh1--pH 2.5 acidphosphatase fusion fragments SEQ ID Nos. :13: and :14: and :15:!.

FIG. 12. Construction of the plasmid pALK533. The pH 2.5 acidphosphatase gene with its own signal sequence was fused to the cbh1promoter.

FIG. 13. Construction of the plasmid pALK532. The pH 2.5 acidphosphatase gene was fused to the cbhI signal sequence and promoter.

FIG. 14. Western blot of the Trichoderma transformants producing pH 2.5acid phosphatase. Lane 1: 10 ng of purified Aspergillus ALKO 243 pH 2.5acid phosphatase; Lane 2: 10 ng of endoF treated Aspergillus ALKO 243 pH2.5 acid phosphatase; and Lanes 3-9: 60ng of protein from the each ofthe culture supernatants of Trichoderma reesei ALKO 2221 transformantsSC-9, KA-31, KA-17, KB-44, KB-18, SB-4 and KA-28, respectively.

DETAILED DESCRIPTION OF THE INVENTION

I. DEFINITIONS

In the description that follows, a number of terms used in recombinantDNA (rDNA) technology are extensively utilized. In order to provide aclear and consistent understanding of the specification and claims,including the scope to be given such terms, the following definitionsare provided.

Gene. A DNA sequence containing a template for an RNA polymerase. TheRNA transcribed from a gene may or may not code for a protein. RNA thatcodes for a protein is termed messenger RNA (mRNA) and, in eukaryotes,is transcribed by RNA polymerase II. A gene containing a RNA polymeraseII template (as a result of a RNA polymerase II promoter) wherein an RNAsequence is transcribed which has a sequence complementary to that of aspecific mRNA, but is not normally translated may also be constructed.Such a gene construct is herein termed an "antisense RNA gene" and suchan RNA transcript is termed an "antisense RNA." Antisense RNAs are notnormally translatable due to the presence of translational stop codonsin the antisense RNA sequence.

A "complementary DNA" or "cDNA" gene includes recombinant genessynthesized by, for example, reverse transcription of mRNA, thus lackingintervening sequences (introns). Genes clones from genomic DNA may ormay not contain introns.

Cloning vehicle. A plasmid or phage DNA or other DNA sequence which isable to carry genetic information, specifically DNA, into a host cell. Acloning vehicle is often characterized by one or a small number ofendonuclease recognition sites at which such DNA sequences may be cut ina determinable fashion without loss of an essential biological functionof the vehicle, and into which a desired DNA may be spliced in order tobring about its cloning into the host cell. The cloning vehicle mayfurther contain a marker suitable for use in the identification of cellstransformed with the cloning vehicle, and origins of replication thatallow for the maintenance and replication of the vehicle in one or moreprokaryotic or eukaryotic hosts. Markers, for example, are tetracyclineresistance or ampicillin resistance. The word "vector" is sometimes usedfor "cloning vehicle." A "plasmid" is a cloning vehicle, generallycircular DNA, that is maintained and replicates autonomously in at leastone host cell.

Expression vehicle. A vehicle or vector similar to a cloning vehicle butwhich supports expression of a gene that has been cloned into it, aftertransformation into a host. The cloned gene is usually placed under thecontrol of (i.e., operably linked to) certain control sequences such aspromoter sequences, that may be provided by the vehicle or by therecombinant construction of the cloned gene. Expression controlsequences will vary depending on whether the vector is designed toexpress the operably linked gene in a prokaryotic or eukaryotic host andmay additionally contain transcriptional elements such as enhancerelements (upstream activation sequences) and termination sequences,and/or translational initiation and termination sites.

Host. A host is a cell, prokaryotic or eukaryotic, that is utilized asthe recipient and carrier of recombinant material.

Eukaryotic host. A "eukaryotic host" may be any cell from a eukaryoticorganism, including, for example, animal, plant, fungi and yeast.

Host of the Invention. The "host of the invention" is a filamentousfungus host that has been engineering to produce recombinant phytaseand/or pH 2.5 acid phosphatase according to the methods of theinvention.

Functional Derivative. A "functional derivative" of a protein or nucleicacid, is a molecule that has been chemically or biochemically derivedfrom (obtained from) such protein or nucleic acid and which retains abiological activity (either functional or structural) that is acharacteristic of the native protein or nucleic acid. The term"functional derivative" is intended to include "fragments," "variants,""analogues," or "chemical derivatives" of a molecule that retain adesired activity of the native molecule.

As used herein, a molecule is said to be a "chemical derivative" ofanother molecule when it contains additional chemical moieties notnormally a part of the molecule. Such moieties may improve themolecule's solubility, absorption, biological half life, etc. Themoieties may decrease the toxicity of the molecule, or eliminate orattenuate any undesirable side effect of the molecule, etc. Moietiescapable of mediating such effects are disclosed in Remington'sPharmaceutical Sciences (1980). Procedures for coupling such moieties toa molecule are well known in the art.

Fragment. A "fragment" of a molecule such as a protein or nucleic acidis meant to refer to a portion of the native amino acid or nucleotidegenetic sequence, and in particular the functional derivatives of theinvention.

Variant or Analog. A "variant" or "analog" of a protein or nucleic acidis meant to refer to a molecule substantially similar in structure andbiological activity to either the native molecule, such as that encodedby a functional allele.

II. THE HOSTS OF THE INVENTION

T. reesei does not produce endogenous phytase. Instead, other enzymecomponents such as β-glucan degrading activity, important in e.g. feedapplications, are produced in high amounts. Thus the use of T. reesei asa production host for fungal phytase and pH 2.5 acid phosphatase resultsin secretion of a totally different enzyme composition when compared tothat secreted from Aspergillus. In addition, by using Trichoderma as asource of a composition containing phytase degrading enzymes, somedifficult problems in downstream processing that occur with similarAspergillus compositions (e.g., in filtration) are avoided. This isbecause the mode of growth of the recombinant T. reesei is differentthan that of Aspergilli, the mycelium being most often fluid and easilyseparable. Thus, by producing these enzymes in the hosts of theinvention, no problems in subsequent filtration of the secreted materialis seen, as is the case with the often slimy and thick mycelium ofAspergilli.

Improved amounts of phytase and pH 2.5 acid phosphatase (as compared tosynthesis in Aspergillus) can be produced in the T. reesei expressionsystem by inserting a DNA sequence obtained from A. niger, coding forphytase or pH 2.5 acid phosphatase activity, into a T. reesei expressioncassette containing the cbh1 promoter and the Aspergillus amdS gene as atransformation marker. Transformation of the construct to T. reeseihosts results in stable transformants expressing the phytase or pH 2.5acid phosphatase in high amounts in a novel background of accompanyingenzyme activities.

The mixture produced by T. reesei contains high β-glucanase activity andlow glucoamylase activity. Moreover, the amount of phytase produced byrecombinant T. reesei strains in shake flask cultivations is comparableto the level of which the main cellulase, the endogenouscellobiohydrolase I, is expressed. The amount of the pH 2.5 acidphosphatase produced by the recombinant strains in shake flaskcultivations is less than 0.5 g/l.

Aspergillus niger var. awamori ALKO 243 (ATCC 38854) (IFO4033) phytaseand acid phosphatase (optimum pH 2.5) were overexpressed in Trichodermareesei under the control of the Trichoderma cellobiohydrolase 1 (cbh1)promoter. In addition, the phytase gene was expressed from its ownpromoter.

For both the genes, two constructions utilizing the cbh1 promoter weremade: in one construction the phytase or acid phosphatase signalsequence was used and in the other construction the cbh1 signal sequencewas used. In all cases, the fusions were made precise by using PCR andthe plasmids were constructed so that the expression cassette could beseparated from the vector backbone prior to transformations. Thus it waspossible to transform strains with only the desired sequences (and notthe entire vector used for maintaining the sequences) and thus to obtainstrains that did not contain any "foreign" sequences; such strains weresuitable for industrial purposes.

Three Trichoderma reesei strains, ATCC 56765 (RutC-30), ALKO 233(VTT-D-79125) and a low aspartyl protease producing strain ALKO 2221were used as hosts for phytase expression. For acid phosphataseexpression, only T. reesei ALKO 2221 was transformed. When phytase wasexpressed under the cbh1 promoter in Trichoderma, the besttransformation with no E. coli sequences produced in shake flaskcultivations about 3,600 fold more phytase than the nontransformed A.niger ALKO 243. When the phytase promoter was used, the best yieldobtained in shake flask cultivations of T. reesei transformants wasabout 120 fold that obtained with A. niger ALKO 243. The best acidphosphatase activities obtained were about 240 fold higher compared tothe levels produced by the A. niger ALKO 243 strain.

The molecular weights (in SDS-PAGE) of the phytase and pH 2.5 acidphosphatase secreted by Trichoderma were different from those secretedby Aspergillus. The difference seemed to be due to differentglycosylation.

The production level of phytase obtained when the Aspergillus gene wasexpressed in Trichoderma under the control of a Trichoderma promoter wassurprisingly high.

The use of T. reesei as a production host for fungal phytase and pH 2.5acid phosphatase results in totally different enzyme preparations ascompared to that from Aspergillus. When compared to Aspergilluspreparations, the mixtures produced by T. reesei contain substantiallyhigher β-glucanase and proportionally lower glucoamylase activities thusmaking T. reesei preparations preferable to be used e.g. in animal feed.

The hosts of the invention are meant to include all Trichoderma.Trichoderma are classified on the basis of morphological evidence ofsimilarity. T. reesei was formerly known as T. viride Pers. or T.koningii Oudem; sometimes it was classified as a distinct species of theT. longibrachiatum group. The entire genus Trichoderma, in general, ischaracterized by rapidly growing colonies bearing tufted or pustulate,repeatedly branched conidiophores with lageniform phialides and hyalineor green conidia borne in slimy heads (Bissett, J., Can. J. Bot.62:924-931 (1984)).

The fungus called T. reesei is clearly defined as a genetic familyoriginating from the strain QM6a, that is, a family of strainspossessing a common genetic background originating from a single nucleusof the particular isolate QM6a. Only those strains are called T. reesei.

Classification by morphological means is problematic and the firstrecently published molecular data from DNA-fingerprint analysis and thehybridization pattern of the cellobiohydrolase-2 (cbh2) gene in T.reesei and T. longibrachiatum clearly indicates a differentiation ofthese strains (Meyer, W. et al., Curr. Genet. 21:27-30 (1992); Morawetz,R. et al., Curr. Genet. 21:31-36 (1992)).

However, there is evidence of similarity between different Trichodermaspecies at the molecular level that is found in the conservation ofnucleic acid and amino acid sequences of macromolecular entities sharedby the various Trichoderma species. For example, Cheng, C., et al.,Nucl. Acids. Res. 18:5559 (1990), discloses the nucleotide sequence ofT. viride cbh1. The gene was isolated using a probe based on the T.reesei sequence. The authors note that there is a 95% homology betweenthe amino acid sequences of the T. viride and T. reesei gene. Goldman,G. H. et al., Nucl. Acids Res. 18:6717 (1990), discloses the nucleotidesequence of phosphoglycerate kinases from T. viride and notes that thededuced amino acid sequence is 81% homologous with the phosphoglyceratekinase gene from T. reesei. Thus, the species classified to T. virideand T. reesei must genetically be very close to each other.

In addition, there is a high similarity of transformation conditionsamong the Trichoderma. Although practically all the industriallyimportant species of Trichoderma can be found in the formerly discussedTrichoderma section Longbrachiatum, there are some other species ofTrichoderma that are not assigned to this section. Such a species is,for example, Trichoderma harzianum, which acts as a biocontrol agentagainst plant pathogens. A transformation system has also been developedfor this Trichoderma species (Herrera-Estrella, A. et al., Molec.Microbiol. 4:839-843 (1990), that is essentially the same as that taughtin the application. Thus, even though Trichoderma harzianum is notassigned to the section Longibrachiatum, the method used byHerrera-Estrella in the preparation of spheroplasts beforetransformation is the same. The teachings of Herrera-Estrella show thatthere is not a significant diversity of Trichoderma spp. such that thetransformation system of the invention would not be expected to functionin all Trichoderma.

Further, there is a common functionality of fungal transcriptionalcontrol signals among fungal species. At least three A. nidulanspromoter sequences, amdS, argB, and gpd, have been shown to give rise togene expression in T. reesei. For amdS and argB, only one or two copiesof the gene are sufficient to being about a selectable phenotypes(Penttila et al., Gene 61:155-164 (1987), Gruber, F. et al., Curr.Genetic 18:71-76 (1990) also notes that that fungal genes can often besuccessfully expressed across different species.

Many species of Trichoderma are available from a wide variety ofresource centers that contain fungal culture collections. In addition,Trichoderma species are catalogued in various databases. These resourcesand databases are summerized by O'Donnell, K. et al., in Biochemistry ofFilamentous Fungi: Technology and Products, D. B. Finkelstein et al.,eds., Butterworth-Heinemann, Stoneham, Mass., USA, 1992, pp. 3-39.

III. CONSTRUCTION OF THE HOSTS OF THE INVENTION

The process for genetically engineering the hosts of the invention,according to the invention, is facilitated through the isolation andpartial sequencing of pure protein encoding an enzyme of interest or bythe cloning of genetic sequences which are capable of encoding suchprotein with polymerase chain reaction technologies; and through theexpression of such genetic sequences. As used herein, the term "geneticsequences" is intended to refer to a nucleic acid molecule (preferablyDNA). Genetic sequences which are capable of encoding a protein arederived from a variety of sources. These sources include genomic DNA,cDNA, synthetic DNA, and combinations thereof. The preferred source ofgenomic DNA is a fungal genomic library. The preferred source of thecDNA is a cDNA library prepared from fungal mRNA grown in conditionsknown to induce expression of the desired mRNA or protein.

The genomic DNA of the invention may or may not include naturallyoccurring introns. Moreover, such genomic DNA may be obtained inassociation with the 5' promoter region of the gene sequences and/orwith the 3' transcriptional termination region. Further, such genomicDNA may be obtained in association with the genetic sequences whichencode the 5' non-translated region of the mRNA and/or with the geneticsequences which encode the 3' non-translated region. To the extent thata host cell can recognize the transcriptional and/or translationalregulatory signals associated with the expression of the mRNA andprotein, then the 5' and/or 3' non-transcribed regions of the nativegene, and/or, the 5' and/or 3' non-translated regions of the mRNA may beretained and employed for transcriptional and translational regulation.Genomic DNA can be extracted and purified from any host cell, especiallya fungal host cell, which naturally expresses the desired protein bymeans well known in the art.

For cloning into a vector, such suitable DNA preparations (eithergenomic DNA or cDNA) are randomly sheared or enzymatically cleaved,respectively, and ligated into appropriate vectors to form a recombinantgene (either genomic or cDNA) library.

A DNA sequence encoding a desired protein or its functional derivativesmay be inserted into a DNA vector in accordance with conventionaltechniques, including blunt-ending or staggered-ending termini forligation, restriction enzyme digestion to provide appropriate termini,filling in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and ligation with appropriateligases. Techniques for such manipulations are disclosed by Maniatis,T., (Maniatis, T. et al., Molecular Cloning (A Laboratory Manual), ColdSpring Harbor Laboratory, second edition, 1988) and are well known inthe art.

Libraries containing sequences coding for the desired gene may bescreened and the desired gene sequence identified by any means whichspecifically selects for a sequence coding for such gene or protein suchas, for example, a) by hybridization with an appropriate nucleic acidprobe(s) containing a sequence specific for the DNA of this protein, orb) by hybridization-selected translational analysis in which native mRNAwhich hybridizes to the clone in question is translated in vitro and thetranslation products are further characterized, or, c) if the clonedgenetic sequences are themselves capable of expressing mRNA, byimmunoprecipitation of a translated protein product produced by the hostcontaining the clone.

Oligonucleotide probes specific for a certain protein which can be usedto identify clones to this protein can be designed from the knowledge ofthe amino acid sequence of the protein or from the knowledge of thenucleic acid sequence of the DNA encoding such protein or a relatedprotein. Alternatively, antibodies may be raised against purified formsof the protein and used to identify the presence of unique proteindeterminants in transformants that express the desired cloned protein.The sequence of amino acid residues in a peptide is designated hereineither through the use of their commonly employed three-letterdesignations or by their single-letter designations. A listing of thesethree-letter and one-letter designations may be found in textbooks suchas Biochemistry, Lehninger, A., Worth Publishers, New York, N.Y. (1970).When the amino acid sequence is listed horizontally, unless otherwisestated, the amino terminus is intended to be on the left end and thecarboxy terminus is intended to be at the right end. Similarly, unlessotherwise stated or apparent from the context, a nucleic acid sequenceis presented with the 5' end on the left.

Because the genetic code is degenerate, more than one codon may be usedto encode a particular amino acid (Watson, J. D., In: Molecular Biologyof the Gene, 3rd Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1977),pp. 356-357). The peptide fragments are analyzed to identify sequencesof amino acids which may be encoded by oligonucleotides having thelowest degree of degeneracy. This is preferably accomplished byidentifying sequences that contain amino acids which are encoded by onlya single codon.

Although occasionally an amino acid sequence may be encoded by only asingle oligonucleotide sequence, frequently the amino acid sequence maybe encoded by any of a set of similar oligonucleotides. Importantly,whereas all of the members of this set contain oligonucleotide sequenceswhich are capable of encoding the same peptide fragment and, thus,potentially contain the same oligonucleotide sequence as the gene whichencodes the peptide fragment, only one member of the set contains thenucleotide sequence that is identical to the exon coding sequence of thegene. Because this member is present within the set, and is capable ofhybridizing to DNA even in the presence of the other members of the set,it is possible to employ the unfractionated set of oligonucleotides inthe same manner in which one would employ a single oligonucleotide toclone the gene that encodes the peptide.

Using the genetic code, one or more different oligonucleotides can beidentified from the amino acid sequence, each of which would be capableof encoding the desired protein. The probability that a particularoligonucleotide will, in fact, constitute the actual protein encodingsequence can be estimated by considering abnormal base pairingrelationships and the frequency with which a particular codon isactually used (to encode a particular amino acid) in eukaryotic cells.Using "codon usage rules," a single oligonucleotide sequence, or a setof oligonucleotide sequences, that contain a theoretical "most probable"nucleotide sequence capable of encoding the protein sequences isidentified.

The suitable oligonucleotide, or set of oligonucleotides, which iscapable of encoding a fragment of a certain gene (or which iscomplementary to such an oligonucleotide, or set of oligonucleotides)may be synthesized by means well known in the art (see, for example,Synthesis and Application of DNA and RNA, S. A. Narang, ed., 1987,Academic Press, San Diego, Calif.) and employed as a probe to identifyand isolate a clone to such gene by techniques known in the art.Techniques of nucleic acid hybridization and clone identification aredisclosed by Maniatis, T., et al., in: Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.(1982), and by Hames, B. D., et al., in: Nucleic Acid Hybridization, APractical Approach, IRL Press, Washington, D.C. (1985). Those members ofthe above-described gene library which are found to be capable of suchhybridization are then analyzed to determine the extent and nature ofcoding sequences which they contain.

To facilitate the detection of a desired DNA coding sequence, theabove-described DNA probe is labeled with a detectable group. Suchdetectable group can be any material having a detectable physical orchemical property. Such materials have been well-developed in the fieldof nucleic acid hybridization and in general most any label useful insuch methods can be applied to the present invention. Particularlyuseful are radioactive labels, such as ³² P, ³ H, ¹⁴ C, 35S, ¹²⁵ I orthe like. Any radioactive label may be employed which provides for anadequate signal and has a sufficient half-life. If single stranded, theoligonucleotide may be radioactively labelled using kinase reactions.Alternatively, polynucleotides are also useful as nucleic acidhybridization probes when labeled with a non-radioactive marker such asbiotin, an enzyme or a fluorescent group.

Thus, in summary, the elucidation of a partial protein sequence, permitsthe identification of a theoretical "most probable" DNA sequence, or aset of such sequences, capable of encoding such a peptide. Byconstructing an oligonucleotide complementary to this theoreticalsequence (or by constructing a set of oligonucleotides complementary tothe set of "most probable" oligonucleotides), one obtains a DNA molecule(or set of DNA molecules), capable of functioning as a probe(s) for theidentification and isolation of clones containing a gene.

In an alternative way of cloning a gene, a library is prepared using anexpression vector, by cloning DNA or, more preferably cDNA prepared froma cell capable of expressing the protein into an expression vector. Thelibrary is then screened for members which express the desired protein,for example, by screening the library with antibodies to the protein.

The above discussed methods are, therefore, capable of identifyinggenetic sequences which are capable of encoding a protein orbiologically active or antigenic fragments of this protein. In order tofurther characterize such genetic sequences, and, in order to producethe recombinant protein, it is desirable to express the proteins whichthese sequences encode. Such expression identifies those clones whichexpress proteins possessing characteristics of the desired protein. Suchcharacteristics may include the ability to specifically bind antibody,the ability to elicit the production of antibody which are capable ofbinding to the native, non-recombinant protein, the ability to provide aenzymatic activity to a cell that is a property of the protein, and theability to provide a non-enzymatic (but specific) function to arecipient cell, among others.

A DNA sequence may be shortened by means known in the art to isolate adesired gene from a chromosomal region that contains more informationthan necessary for the utilization of this gene in the hosts of theinvention. For example, restriction digestion may be utilized to cleavethe full-length sequence at a desired location. Alternatively, or inaddition, nucleases that cleave from the 3'-end of a DNA molecule may beused to digest a certain sequence to a shortened form, the desiredlength then being identified and purified by gel electrophoresis and DNAsequencing. Such nucleases include, for example, Exonuclease III andBal31. Other nucleases are well known in the art.

If the coding sequence and an operably linked promoter are introducedinto a recipient eukaryotic cell as a non-replicating DNA (or RNA),non-integrating molecule, the expression of the encoded protein mayoccur through the transient (nonstable) expression of the introducedsequence.

Preferably the coding sequence is introduced on a DNA (or RNA) molecule,such as a closed covalent circular molecule that is incapable ofautonomous replication, or preferable a linear molecule that integratesinto the host chromosome. Genetically stable transformants may beconstructed with vector systems, or transformation systems, whereby adesired DNA is integrated into the host chromosome. Such integration mayoccur de novo within the cell or, be assisted by transformation with avector which functionally inserts itself into the host chromosome, forexample, transposons or other DNA elements which promote integration ofDNA sequences in chromosomes. A vector is employed which is capable ofintegrating the desired gene sequences into a fungal host cellchromosome.

The genes coding for phytase or pH 2.5 acid phosphatase under thecontrol of suitable promoters may be combined in one plasmidconstruction and introduced into the host cells by transformation. Thenature of the plasmid vector will depend on the host organism. In thepractical realization of the invention the filamentous fungusTrichoderma has been employed as a model. Thus, for Trichoderma andespecially for T. reesei, vectors incorporating DNA that provides forintegration of the sequences encoding the phytase or pH 2.5 acidphosphatase genes into the host's chromosome are preferred. Targetingthe integration to the cbh1 (DNA encoding the enzyme cellobiohydrolaseI) locus of the host is the preferred method of obtaining the high levelexpression of the phytase or pH 2.5 acid phosphatase genes of theinvention, such targeting may be achieved by providing cbh1 coding orflanking sequences on the recombinant construct, in an amount sufficientto direct integration to this locus at a relevant frequency.

Cells which have stably integrated the introduced DNA into theirchromosomes are selected by also introducing one or more markers whichallow for selection of host cells which contain the expression vector inthe chromosome, for example the marker may provide biocide resistance,e.g., resistance to antibiotics, or heavy metals, such as copper, or thelike. The selectable marker gene can either be directly linked to theDNA gene sequences to be expressed, or introduced into the same cell byco-transfection. A genetic marker especially for the transformation ofthe hosts of the invention is amdS, encoding acetamidase and thusenabling Trichoderma to grow on acetamide as the only nitrogen source.

To express a desired protein and/or its active derivatives,transcriptional and translational signals recognizable by an appropriatehost are necessary. The cloned coding sequences, obtained through themethods described above, and preferably in a double-stranded form, maybe operably linked to sequences controlling transcriptional expressionin an expression vector, and introduced into a host cell, eitherprokaryote or eukaryote, to produce recombinant protein or a functionalderivative thereof. Depending upon which strand of the coding sequenceis operably linked to the sequences controlling transcriptionalexpression, it is also possible to express antisense RNA or a functionalderivative thereof.

Expression of the protein in different hosts may result in differentpost-translational modifications which may alter the properties of theprotein. Preferably, the present invention encompasses the expression ofthe protein or a functional derivative thereof, in eukaryotic cells, andespecially in fungus.

A nucleic acid molecule, such as DNA, is said to be "capable ofexpressing" a polypeptide if it contains expression control sequenceswhich contain transcriptional regulatory information and such sequencesare "operably linked" to the nucleotide sequence which encodes thepolypeptide.

An operable linkage is a linkage in which a sequence is connected to aregulatory sequence (or sequences) in such a way as to place expressionof the sequence under the influence or control of the regulatorysequence. Two DNA sequences (such as a coding sequence and a promoterregion sequence linked to the 5' end of the coding sequence) are said tobe operably linked if induction of promoter function results in thetranscription of mRNA encoding the desired protein and if the nature ofthe linkage between the two DNA sequences does not (1) result in theintroduction of a frame-shift mutation, (2) interfere with the abilityof the expression regulatory sequences to direct the expression of theprotein, antisense RNA, or (3) interfere with the ability of the DNAtemplate to be transcribed. Thus, a promoter region would be operablylinked to a DNA sequence if the promoter was capable of effectingtranscription of that DNA sequence.

The precise nature of the regulatory regions needed for gene expressionmay vary between species or cell types, but shall in general include, asnecessary, 5' non-transcribing and 5' non-translating (non-coding)sequences involved with initiation of transcription and translationrespectively, such as the TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5' non-transcribing control sequences willinclude a region which contains a promoter for transcriptional controlof the operably linked gene. Such transcriptional control sequences mayalso include enhancer sequences or upstream activator sequences, asdesired.

Expression of a protein in eukaryotic hosts such as fungus requires theuse of regulatory regions functional in such hosts, and preferablyfungal regulatory systems. A wide variety of transcriptional andtranslational regulatory sequences can be employed, depending upon thenature of the host. Preferably, these regulatory signals are associatedin their native state with a particular gene which is capable of a highlevel of expression in the host cell.

In eukaryotes, where transcription is not linked to translation, suchcontrol regions may or may not provide an initiator methionine (AUG)codon, depending on whether the cloned sequence contains such amethionine. Such regions will, in general, include a promoter regionsufficient to direct the initiation of RNA synthesis in the host cell.Promoters from filamentous fungal genes which encode a mRNA productcapable of translation are preferred, and especially, strong promoterscan be employed provided they also function as promoters in the hostcell. Preferred strong eukaryotic promoters for use in Trichodermainclude the T. reesei cbh1 gene promoter or a promoter of anothercellulase gene such as that for the cbh2, eg/1 or eg/2 gene may be used.In addition to the use of Trichoderma regulatory elements, theexpression of proteins may be placed under the control of regulatoryelements from Aspergillus nidulans (for example, the argB gene promoterand the amdS gene promoter), Aspergillus niger (for example, the phytasepromoter or the glucoamylase gene promoter) However, expression undernon-Trichoderma regulatory elements such as these may be very low ascompared to the use of Trichoderma elements, and especially those of T.reesei.

As is widely known, translation of eukaryotic mRNA is initiated at thecodon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the desired protein, or a functionalderivative thereof, does not contain any intervening codons which arecapable of encoding a methionine. The presence of such codons resultseither in a formation of a fusion protein (if the AUG codon is in thesame reading frame as the protein-coding DNA sequence) or a frame-shiftmutation (if the AUG codon is not in the same reading frame as theprotein-coding sequence).

It may be desired to construct a fusion product that contains a partialcoding sequence (usually at the amino terminal end) of a protein and asecond coding sequence (partial or complete) of a phytase degradingenzyme of the invention. The sequence that does not encode the phytasedegrading enzyme may or may not function as a signal sequence forsecretion of the protein from the host cell. For example, the sequencecoding for desired protein may be linked to a signal sequence which willallow secretion of the protein from, or the compartmentalization of theprotein in, a particular host. Such fusion protein sequences may bedesigned with or without specific protease sites such that a desiredpeptide sequence is amenable to subsequent removal. In a preferredembodiment, the native signal sequence of a fungal protein is used, or afunctional derivative of that sequence that retains the ability todirect the secretion of the peptide that is operably linked to it.Aspergillus leader/secretion signal elements also function inTrichoderma.

Transcriptional initiation regulatory signals can be selected whichallow for repression or activation, so that expression of the operablylinked genes can be modulated. For example, regulatory signals may betemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or are subject to chemical regulation, e.g.,metabolite. Translational signals are not necessary when it is desiredto express antisense RNA sequences.

If desired, the non-transcribed and/or non-translated regions 3' to thesequence coding for a desired protein can be obtained by theabove-described cloning methods. The 3'-non-transcribed region may beretained for its transcriptional termination regulatory sequenceelements, or for those elements which direct polyadenylation ineukaryotic cells. Where the native expression control sequences signalsdo not function satisfactorily in a host cell, then sequences functionalin the host cell may be substituted.

The vectors of the invention may further comprise other operably linkedregulatory elements such as DNA elements which confer antibioticresistance, or origins of replication for maintenance of the vector inone or more host cells.

In another embodiment, especially for maintenance of the vectors of theinvention in prokaryotic cells, or in yeast S. cerevisiae cells, theintroduced sequence is incorporated into a plasmid or viral vectorcapable of autonomous replication in the recipient host. Any of a widevariety of vectors may be employed for this purpose. In Bacillus hosts,integration of the desired DNA may be necessary.

Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to"shuttle" the vector between host cells of different species.

Preferred S. cerevisiae yeast plasmids include those containing the2-micron circle, etc., or their derivatives. Such plasmids are wellknown in the art (Botstein, D., et al., Miami Wntr. Symp. 19:265-274(1982); Broach, J. R., in: The Molecular Biology of the YeastSaccharomyces: Life Cycle and Inheritance, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach, J. R.,Cell 28:203-204 (1982); Bollon, D. P., et al., J. Clin. Hematol. Oncol.10:39-48 (1980); Maniatis, T., In: Cell Biology: A ComprehensiveTreatise, Vol. 3, Gene Expression, Academic Press, N.Y., pp. 563-608(1980)), and are commercially available.

Once the vector or DNA sequence containing the construct(s) is preparedfor expression, the DNA construct(s) is introduced into an appropriatehost cell by any of a variety of suitable means, includingtransformation. After the introduction of the vector, recipient cellsare grown in a selective medium, which selects for the growth ofvector-containing cells. Expression of the cloned gene sequence(s)results in the production of the desired protein, or in the productionof a fragment of this protein. This expression can take place in acontinuous manner in the transformed cells, or in a controlled manner,for example, by induction of expression.

Fungal transformation is carried out also accordingly to techniquesknown in the art, for example, using, for example, homologousrecombination to stably insert a gene into the fungal host and/or todestroy the ability of the host cell to express a certain protein.

IV. PREPARATION OF ANTIBODIES

In the following description, reference will be made to variousmethodologies well-known to those skilled in the art of immunology.Standard reference works setting forth the general principles ofimmunology include the work of Catty, D. (Antibodies, A PracticalApproach, Vol. 1, IRL Press, Washington, D.C. (1988)); Klein, J.(Immunology: The Science of Cell-Noncell Discrimination, John Wiley &Sons, New York (1982)); Kennett, R., et al. in Monoclonal Antibodies,Hybridoma: A New Dimension in Biological Analyses, Plenum Press, NewYork (1980); Campbell, A. ("Monoclonal Antibody Technology," in:Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13(Burdon, R., et al., eds.), Elsevier, Amsterdam (1984)); and Eisen, H.N., in: Microbiology, 3rd Ed. (Davis, B. D., et al., Harper & Row,Philadelphia (1980)).

An antibody is said to be "capable of binding" a molecule if it iscapable of specifically reacting with the molecule to thereby bind themolecule to the antibody. The term "epitope" is meant to refer to thatportion of a hapten which can be recognized and bound by an antibody. Anantigen may have one, or more than one epitope. An "antigen" is capableof inducing an animal to produce antibody capable of binding to anepitope of that antigen. The specific reaction referred to above ismeant to indicate that the antigen will react, in a highly selectivemanner, with its corresponding antibody and not with the multitude ofother antibodies which may be evolved by other antigens.

The term "antibody" (Ab) or "monoclonal antibody" (Mab) as used hereinis meant to include intact molecules as well as fragments thereof (suchas, for example, Fab and F(ab')₂ fragments) which are capable of bindingan antigen. Fab and F(ab')₂ fragments lack the Fc fragment of intactantibody, clear more rapidly from the circulation, and may have lessnon-specific tissue binding of an intact antibody (Wahl et al., J. Nucl.Med. 24:316-325 (1983)).

The antibodies of the present invention are prepared by any of a varietyof methods. Preferably, purified phytase or pH 2.5 acid phosphataseprotein, or a fragment thereof, (treated or not treated with endoF orits equivalent to remove sugar moieties), is administered to an animalin order to induce the production of sera containing polyclonalantibodies that are capable of binding such phytase or pH 2.5 acidphosphatase.

Cells expressing phytase or pH 2.5 acid phosphatase protein, or afragment thereof, or, a mixture of proteins containing phytase or pH 2.5acid phosphatase or such fragments, can also be administered to ananimal in order to induce the production of sera containing polyclonalantibodies, some of which will be capable of binding phytase or pH 2.5acid phosphatase protein. If desired, such phytase or pH 2.5 acidphosphatase antibody may be purified from the other polyclonalantibodies by standard protein purification techniques and especially byaffinity chromatography with purified phytase or pH 2.5 acid phosphataseor fragments thereof.

A phytase or pH 2.5 acid phosphatase protein fragment may also bechemically synthesized and purified by HPLC to render it substantiallyfree of contaminants. Such a preparation is then introduced into ananimal in order to produce polyclonal antisera of high specificactivity.

Monoclonal antibodies can be prepared using hybridoma technology (Kohleret al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511(1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al.,in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp.563-681 (1981)). In general, such procedures involve immunizing ananimal with phytase or pH 2.5 acid phosphatase protein antigen. Thesplenocytes of such animals are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP₂ O), available from the AmericanType Culture Collection, Rockville, Md. After fusion, the resultinghybridoma cells are selectively maintained in HAT medium, and thencloned by limiting dilution as described by Wands, J. R., et al.,Gastroenterology 80:225-232 (1981), which reference is hereinincorporated by reference. The hybridoma cells obtained through such aselection are then assayed to identify clones which secrete antibodiescapable of binding the phytase or pH 2.5 acid phosphatase proteinantigen.

Through application of the above-described methods, additional celllines capable of producing antibodies which recognize epitopes of thephytase or pH 2.5 acid phosphatase protein can be obtained.

Antibodies against both highly conserved and poorly conserved regions ofthe phytase or pH 2.5 acid phosphatase protein are useful for studies onthe control of biosynthesis and catabolism of phytase or pH 2.5 acidphosphatase protein, and for studies wherein it is necessary to identifyor quantitate the presence and/or of the protein antigen in acomposition.

V. PRODUCTION OF PHYTASE AND ACID PHOSPHATASE

The best phytase production levels are obtained when Trichoderma aretransformed with linear DNA and sing the cbh1 promoter (about 3800PNU/ml, see Table 8). About 3480 and 3600 PNU/ml culture medium wasobtained with the best T. reesei ALKO 2221 and ALKO 233 transformantscontaining no E. coli sequences, respectively. The best T. reesei ATCC56765 transformant with no E. coli sequences produced about 1,800 PNU/mlculture medium. Both the phytase and the cbh1 signal sequence seemed towork equally well. In one host, (ALKO 233), the level of phytase washigher when the phytase signal sequence was used.

Phytase is expressed from the Trichoderma hosts of the invention intothe supernatant of the culture medium. The amount of phytase in theculture medium is generally higher than any hitherto reported amount ofa heterologous protein that was expressed in Trichoderma.

The spectrum of enzymes that accompany phytase in the Trichodermastrains of the invention is greatly different and advantageous over thatof similar preparations of Aspergillus culture supernatants. Bothendoglucanase and cellobiohydrolase activities are generallysubstantially higher using the Trichoderma hosts of the invention. Theglycosylation pattern of the phytase is also different when it isexpressed from Trichoderma, resulting in a phytase protein that migratesas multiple bands on Western analysis.

The best production of pH 2.5 acid phosphatase from the Trichodermatransformants of the invention resulted in 240 APNU/ml culture medium,in shake flask cultivation and in lactose based medium. As with thephytase, both the acid phosphatase and the cbh1 signal sequence workedequally well.

The compositions of the invention that contain phytase may be useddirectly for the removal of phytic acid, or inositol hexaphosphoricacid, from raw material, especially phytin-containing raw material, andespecially plant material. Phytase removes the phosphate groups fromphytic acid and destroys its ability to interfere with mineralabsorption. When used as an animal feed additive, the phytasecompositions of the invention release phosphate bound to phytin in grainand thus dramatically reduce the need for doses of additional phosphatein feed formulations and lessen environmental loads.

The phytase and pH 2.5 acid phosphatase produced according to theinvention may be purified by protein purification methods known in theart.

Having now generally described the invention, the same will becomebetter understood by reference to certain specific examples that areincluded herein for purposes of illustration only and are not intendedto be limiting unless otherwise specified.

EXAMPLES

Example 1

Method of Assay of Phytase Activity

Principle Phytase acts on phytate (inositol hexaphosphate) to releaseinorganic phosphate. The determination of released inorganic phosphateis based on the color formed by the reduction of a phosphomolybdatecomplex.

Unit of Activity One phytase unit (PU) is the amount of enzyme whichliberates, under standard conditions, 1 nmol of inorganic phosphate fromsodium phytate in one minute.

Assay conditions

Substrate Sodium phytate

pH 5.0

incubation temperature 37° C. ±0.5° C.

incubation time 15 minutes

Equipment

Water bath 37° C.

Water bath 50° C.

Spectrophotometer

Test tube mixer (vortex)

Phosphate Free Glassware

Reagents All solutions are prepared in deionized water, Milli-Q orequivalent.

1. Citrate Buffer (0.2M, pH 5.0). Prepare 0.2M solutions of both sodiumcitrate (C₆ H₈ O₇ Na.2H₂ O, 58.8 g/l, Merck 6448) and citric acid (C₆ H₈O₇.H₂ O, 42.0 g/l, Merck 244) in water. Adjust the pH of the citratesolution (1 liter) to 5.0 with 0.2M citric acid (the consumption ofcitric acid solution should be about 385 ml).

2. Substrate. Dissolve 1.00 g of sodium phytate (Sigma P-3168) in about70 ml citrate buffer. Adjust the pH to 5.0 with 0.2M citric acid andadjust the volume to 100 ml with citrate buffer. Fresh substratesolution must be prepared daily.

3. 15% (w/v) TCA Solution. Prepare from trichloroacetic acid (Merck807).

4. 10% (w/v) Ascorbic Acid Solution. Prepare from ascorbic acid (Merck127). Store under refrigeration. The solution is stable for seven days.

5. 2.5 % (w/v) Ammonium Molybdate Solution. Dissolve 2.5 g (NH₄)₆ Mo₇O₂₄.4H₂ O, Merck 1182) in water and make up to 100 ml.

6. 1M Sulfuric Acid. Add 55.6 ml of concentrated H₂ SO₄ (Merck 731) toabout 800 ml of water, with stirring. Allow to cool and make up to 1000ml with water.

7. Reagent C. Mix 3 volumes of 1M sulfuric acid with 1 volume of 2.5%ammonium molybdate, then add 1 volume of 10% ascorbic acid and mix well.Fresh reagent C must be prepared daily.

Sample dilution Samples are diluted in citrate buffer. Make duplicatedilutions of each sample. In case of enzyme powder weigh accuratelyabout 250 mg of sample, dissolve in the buffer and fill to 25 ml in avolumetric flask, dilute further if necessary.

    ______________________________________                                        Dilution table:                                                               Estimated activity                                                            PU/ml        Recommended dilution                                                                        Dilution factor                                    ______________________________________                                         2000        1 + 19         20                                                 20000       1 + 199        200                                                40000       1 + 399        400                                               100000       1 + 999       1000                                               500000        1 + 4999     5000                                               ______________________________________                                    

Assay

Hydrolysis Pipette 1.0 ml of sample dilution containing 20-190 PU in twotest tubes. Add 2.0 ml of 15% TCA to one of the tubes (blank) and mix.Put the tubes without TCA in a water bath at 37° C. and let themequilibrate for 5 minutes. Using a stopwatch start the hydrolysis byadding sequentially at proper intervals 1.0 ml of substrate(equilibrated for about 10 minutes at 37° C.) to each tube and mix.After exactly 15 minutes incubation stop the reaction by adding 2.0 mlof TCA to each tube. Mix and cool to room temperature. Add 1.0 mlsubstrate to the blank tubes (kept at room temperature) also and mix. Ifprecipitate occurs it must be separated by centrifugation for 10 minutesat 2000.g.

Released orthophosphate Pipette 0.4 ml of each sample after hydrolysisin test tubes. Add 3.6 ml of water to each tube. Add 4.0 ml of reagent Cand mix. Incubate at 50° C. for 20 minutes and cool to room temperature.Measure the absorbance against that of reagent blank (see below) at 820nm.

Standard Prepare a 9.0 mM phosphate stock solution. Dissolve and dilute612.4 mg KH₂ PO₄ (Merck 4873, dried in desiccator with silica) to 500 mlwith water in a volumetric flask. Make the following dilutions in waterfrom the stock solution and use these as standards.

    ______________________________________                                                   Phosphorus    Phytase activity                                     Dilution   concentration nmol/ml                                                                       PU/ml*                                               ______________________________________                                        1:100      90            240                                                  1:200      45            120                                                  1:400      22.5           60                                                  ______________________________________                                         *The corresponding phytase activity (PU/ml) is obtained by dividing the       phosphorous concentration (nmol/ml) by the time of hydrolysis (15 minutes     and multiplying by four (total volume after hydrolysis reaction/sample        volume) and by 10 (dilution before analysis of inorganic phosphorous).   

Pipette 4.0 ml of each dilution to two test tubes. Pipette also 4.0 mlof water in one tube (reagent blank). Add 4.0 ml of reagent C and mix.Incubate at 50° C. for 20 minutes and cool to room temperature. Measurethe absorbances at 820 nm against that of reagent blank. Prepare astandard curve by blotting the absorbances against phytase activity(PU/ml). A new standard line must be constructed with each series ofassays.

Calculation Subtract the blank absorbance from the sample absorbance(the difference should be 0.100-1.000). Read the phytase activity(PU/ml) from the standard line and multiply by the dilution factor. Tocalculate the activity (PU/g) of enzyme powders the result (PU/ml) isfurther multiplied by 25 (ml) and divided by the exact weight of thesample (g).

Preparation of feed and other insoluble samples for phytase analysis

Weigh accurately about 2.5 g of ground sample in two 50 ml beakers. Add20.0 ml of citrate buffer. Mix using a magnetic stirrer for 30 minutesat room temperature. Transfer about 10 ml of each in centrifuge tubesand separate the solid matter by centrifugation for 10 minutes at2000.g. Apply 2.5 ml of supernatant on PD-10 gel filtration columns(Sephadex G-25M, Pharmacia 17-0851-01) equilibrated with 25 ml citratebuffer. Discard the eluate. Then apply 3.5 ml citrate buffer on thecolumn and collect the eluate in a graduated cylinder. Fill the volumeto 5.0 ml with citrate buffer (dilution factor 2) and assay for phytaseactivity. The activity PU/g is obtained by multiplying the measuredactivity (PU/ml) by 40 (dilution factor·volume of extraction buffer) anddividing by the exact weight of sample (g). Reference: Chen et al. Anal.Chem. 28:1756-1758 (1956).

Example 2

Assay of Acid Phosphatase Activity

Principle. Acid phosphatase acts on p-nitrophenyl phosphate to releaseinorganic phosphate. The determination of released inorganic phosphateis based on the color formed by the reduction of phosphomolybdatecomplex.

Unit of activity. One acid phosphatase unit (HFU) is the amount ofenzyme which liberates, under standard conditions, 1 nmol of inorganicphosphate from p-nitrophenyl phosphate in one minute.

Assay conditions

Substrate p-nitrophenyl phosphate

pH 2.5

Temperature 37° C. ±0.5° C.

Incubation time 15 min

Equipment

Water bath 37° C.

Water bath 50° C.

Spectrophotometer

Test tube mixer (vortex)

Centrifuge (Hereaus Biofuge

17S, 3090 or equivalent,

Phosphate Free Glassware

Reagents. All solutions are prepared in deionized water, Milli-Q orequivalent.

1. Glycine Buffer (0.2M, pH 2.5)

Dissolve 15.014 g glycine (Merck 4201) in about 800 ml of water. Adjustthe pH to 2.5 with 1M hydrochloric acid (consumption should be about 80ml) and dilute to 1000 ml with water.

2. Substrate (30 mM)

Dissolve 1.114 g p-nitrophenyl phosphate (Boehringer, 738 352) inglycine buffer and adjust the volume to 100 ml with the buffer. Freshsubstrate solution must be prepared daily.

3. 15% (w/v) TCA Solution

Prepare from trichloroacetic acid (Merck 807).

4. 10% (w/v) Ascorbic Acid Solution

Prepare from ascorbic acid (Merck 127). Store under refrigeration. Thesolution is stable for 7 days.

5. 2.5% (w/v) Ammonium Molybdate Solution

Dissolve 2.5 g (NH₄)₆ MO₇ O₂₄.4H₂ O, Merck 1182) in water and make up to100 ml.

6. 1M Sulphuric Acid

Add 55.6 ml of concentrated H₂ SO₄ (Merck 731) to about 800 ml of water,with stirring. Allow to cool and make up to 1000 ml with water.

7. Reagent C

Mix 3 volumes of 1M sulphuric acid with 1 volume of 2.5% ammoniummolybdate, then add 1 volume of 10% ascorbic acid and mix well. Freshreagent C must be prepared daily.

Sample dilution. Samples are diluted in glycine buffer. Make duplicatedilutions of each sample. In case of enzyme powder weigh accuratelyabout 250 mg of sample, dissolve in the buffer and fill to 25 ml in avolumetric flask, dilute further if necessary.

    ______________________________________                                        Dilution table:                                                               Estimated activity                                                                            Recommended                                                                              Dilution                                           HFU/ml          dilution   factor                                             ______________________________________                                         20000          1 + 19      20                                                 200000         1 + 199     200                                                400000         1 + 399     400                                               1000000         1 + 999    1000                                               5000000          1 + 4999  5000                                               ______________________________________                                    

Assay

Hydrolysis: Pipette 1.9 ml of substrate in two test tubes. Add 2.0 ml of15% TCA to one of the tubes (blank) and mix. Put the tubes without TCAin a water bath at 37° C. and let them equilibrate for 5 min. Using astopwatch start the hydrolysis by adding sequentially at properintervals 0.1 ml of enzyme dilution to each tube and mix. After exactly15 min incubation stop the reaction by adding 2.0 ml of TCA to eachtube. Mix and cool to room temperature. Add 0.1 ml of sample to theblank tubes (kept at room temperature) also and mix. If precipitateoccurs it must be separated by centrifugation for 10 min at 2000.g.

Released orthophosphate: Pipette 0.4 ml of each sample after hydrolysisin test tubes. Add 3.6 ml of water to each tube. Add 4.0 ml of reagent Cand mix. Incubate at 50° C. for 20 min and cool to room temperature.Measure the absorbance against that of reagent blank (see below) at 820nm.

Standard. Prepare a 9.0 mM phosphate stock solution. Dissolve and dilute612.4 mg KH₂ PO₄ (Merck 4873, dried in dessicator with silica) to 500 mlwith water in a volumetric flask. Make the following dilutions in waterfrom the stock solution and use these as standards.

    ______________________________________                                                 Phosphorus concentration                                                                     Acid phosphatase activity                             Dilution nmol/ml        HFU/ml*                                               ______________________________________                                        1:100    90             2400                                                  1:200    45             1200                                                  1:400    22.5            600                                                  ______________________________________                                         *The corresponding acid phosphatase activity (HFU/ml) is obtained by          dividing the phosphorus concentration (nmol/ml) by the time of hydrolysis     (15 min) and multiplying by 40 (total volume after hydrolysis                 reaction/sample volume) and by 10 (dilution before analysis of inorganic      phosphorus).                                                             

Pipette 4.0 ml of each dilution to two test tubes. Pipette also 4.0 mlof water in one tube (reagent blank). Add 4.0 ml of reagent C and mix.Incubate at 50° C. for 20 min and cool to room temperature. Measure theabsorbances at 820 nm against that of reagent blank. Prepare a standardcurve by blotting the absorbances against acid phosphatase activity(HFU/ml). A new standard line must be constructed with each series ofassays.

Calculation. Subtract the blank absorbance from the sample absorbance(the difference should be 0.100-1.000). Read the acid phosphataseactivity (HFU/ml) from the standard line and multiply by the dilutionfactor. To calculate the activity (HFU/g) of enzyme powders the result(HFU/ml) is further multiplied by 25 (ml) and divided by the exactweight of the sample (g).

Preparation of feed and other insoluble samples for acid phosphataseanalysis. Weigh accurately about 2.5 g of ground sample in two 50 mlbeakers. Add 20.0 ml of glycine buffer. Mix using a magnetic stirrer for30 min at room temperature. Transfer about 10 ml of each in centrifugetubes and separate the solid matter by centrifugation for 10 min at2000.g. Apply 2.5 ml of supernatant on PD-10 gel filtration columns(Sephadex G-25M, Pharmacia 17-0851-01) equilibrated with 25 ml glycinebuffer. Discard the eluate. Then apply 3.5 ml of glycine buffer on thecolumn and collect the eluate in a graduated cylinder. Fill the volumeto 5.0 ml with glycine buffer (dilution factor 2) and assay for acidphosphatase activity. The activity HFU/g is obtained by multiplying themeasured activity (HFU/ml) by 40 (dilution factor·volume of extractionbuffer) and dividing by the exact weight of sample (g). Reference: Chen,P. S., et al., Anal. Chem. 28:1756-1758 (1956).

Example 3

Purification of Phytase and pH 2.5 Acid Phosphatase

For reference to how the skilled artisan would purify phytase and pH 2.5acid phosphatase, the following are provided.

I. PHYTASE

Enzyme purification. Steps were done at 4° to 8° C. unless otherwisestated. The starting material was the cell-free culture mediumconcentrate produced by Aspergillus niger var. awamori ALKO 243.

Ammonium sulphate precipitation. The culture filtrate concentrate (990ml) was kept on an ice bath and 0.436 g ammonium sulphate per ml wasadded (70% saturation). After 30 minutes the precipitate was separatedby centrifugation for 15 minutes at 10000.g and discarded.

Hydrophobic interaction chromatography. The supernatant (1070 ml) wasapplied to an Octyl-Sepharose CL-4B (Pharmacia) column (5 cm×17 cm)equilibrated with a solution containing 0.436 g (NH₄)₂ SO₄ per ml of 20mM bis-Tris/HCl (pH 6.2). The column was washed with 500 ml of theequilibration solution and then developed with a linear gradient of 500ml containing 70→0% amonium sulfate in 20 mM bis-Tris/HCl (pH 6.2).Fractions of 10 ml were collected and analyzed for phytase and acidphosphatase activity. Most of the phytase activity eluted in thebeginning of the gradient. The fractions were pooled for the next step.The fractions eluting after phytase activity and containing most of theacid phosphatase activity were pooled for acid phosphatase purification(see below).

Anion exchange chromatography. The pooled phytase fractions (129 ml)were concentrated by ultrafiltration using an Amicon PM 10 membrane. Theresidual ammonium sulphate was removed by PD 10 (Pharmacia) gelfiltration columns equilibrated with 50 mM bis-Tris/HCl (pH 6.2). Thesample, in 24.5 ml, was applied to a DEAE-Sepharose (Pharmacia) column(5 cm×7 cm) equilibrated with 50 mM bis-Tris/HCl (pH 6.2). The columnwas washed with the equilibrium buffer (100 ml) and developed by alinear gradient of 200 ml containing 0→0.5M NaCl in equilibrium buffer.

Gel filtration. The pooled active fractions were concentrated using aCentricon -30 microconcentrator to a total volume of 600 μl. Portions of100 μl were run at about 23° C. and 0.3 ml/min through a Superose 12 HR10/30 HPLC column (Pharmacia) equilibrated with 50 mM bis-Tris/HCl (pH6.2).

Cation exchange. The pooled active fractions were transferred to 50 mMsodium formiate (pH 3.8) using a Centricon -30 microconcentrator. Thesample was applied in two portions of 2 ml to a Mono S HR 5/5 FPLCcolumn (Pharmacia) equilibrated with 50 mM sodium formiate (pH 3.8) atabout 23° C. The column was washed with the equilibration buffer (10 ml)and the bound protein was eluted at 60 ml/h with a linear gradient of 20ml containing 0→430 mM NaCl in equilibration buffer.

II. ACID PHOSPHATASE

Gel filtration. The pooled fractions containing most of the acidphosphatase activity from the hydrophobic interaction chromatographystep were concentrated by ultrafiltration using an Amicon PM 10membrane. The concentrated sample (25 ml) was run through a SephacrylS-200 (Pharmacia) column (2.6 cm×94 cm) equilibrated with 50 mMbis-Tris/HCl (pH 6.2) at 20 ml/h.

Anion exchange chromatography. The pooled fractions (48 ml) were appliedto a DEAE-Sepharose (Pharmacia) column (5 cm×7 cm) equilibrated with 50mM bis-Tris/HCl (pH 6.2). The column was washed with 100 ml ofequilibration buffer and developed with a linear gradient of 200 mlcontaining 0→0.5M NaCl in equilibration buffer.

Anion exchange chromatography. The pooled active fractions wereconcentrated and transferred to 20 mM bis-Tris/HCl (pH 6.0) byultrafiltration using an Amicon PM 10 membrane. The sample was run infour portions of 3.5 ml on Mono Q HR 515 HPLC column (Pharmacia)equilibrated with 20 mM bis-Tris/HCl (pH 6.0) at about 23° C. and 60ml/h. The column was washed with 10 ml of the equilibrium buffer and thebound protein was eluted with a linear gradient of 20 ml containing0→350 mM NaCl in equilibrium buffer.

Gel filtration. The active fractions were pooled, concentrated andtransferred to 20 mM bis-Tris/HCl (pH 6.2) containing 150 mM NaCl withCentricon -30 microconcentrator to total volume of 400 μl. Portions of100 μl were run at about 23° C. and 18 ml/h through a Superose 12 HR10/30 HPLC column (Pharmacia) equilibrated with the sample buffer.

Anion exchange chromatography. The pooled active fractions weretransferred to 20 mM histidin/HCl (pH 5.8) with a PD 10 gel filtrationcolumn. The sample was run in four portions of 1 ml on Mono Q HR 5/5HPLC column (Pharmacia) equilibrated with the sample buffer at about 23°C. and 60 ml/h. The column was washed with 5 ml of the sample buffer andthe bound protein was eluted with a linear gradient of 20 ml containing0→350 mM NaCl in equilibrium buffer.

                  TABLE 1                                                         ______________________________________                                        Summary of purification of phytase from Aspergillus niger                             Total    Total    Specific                                                    activity protein  activity                                                                             Yield Purification                           Step    (U)      (mg)     (U/mg) (%)   (fold)                                 ______________________________________                                        Culture 4486680  2119      2117  100   1                                      filtrate                                                                      Ammonium                                                                              3771750  1263      2986  84.1  1.4                                    sulfate                                                                       supernatant                                                                   Octyl   1765881  32.3      54671 39.4  26                                     Sepharose                                                                     DEAE-   1453470  8.4      173032 32.4  82                                     Sepharose                                                                     Superose 12                                                                           1010888  5.7      177349 22.5  84                                     Mono S   827566  3.0      275885 18.4  130                                    ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Summary of purification of acid phosphatase from                              Aspergillus niger                                                                     Total     Total   Specific                                                    activity  protein activity                                                                             Yield Purification                           Step    (U)       (mg)    (U/mg) (%)   (fold)                                 ______________________________________                                        Culture 116523000 2119     54990 100   1                                      filtrate                                                                      Ammonium                                                                              88275000  1263     69893 75.8  1.3                                    sulphate                                                                      supernatant                                                                   Octyl   68296470  583      117147                                                                              58.6  2.1                                    Sepharose                                                                     Sephacryl                                                                             52237600  97.9     533581                                                                              44.8  9.7                                    DEAE-   46127692  54.6     844830                                                                              39.6  15.4                                   Sepharose                                                                     Mono Q  19326753  3.28    5892303                                                                              16.6  107                                    Superose                                                                              16876978  nd      nd     14.5  nd                                     Mono Q  15197050  2.2     6907750                                                                              13.0  126                                    ______________________________________                                         nd = not determined                                                      

Example 4

Characterization of Purified Phytase and pH 2.5 Acid Phosphatase PeptideDigestions

Native purified phytase (70 μg) in 50 mM Tris-HCl pH 7.9 was digestedwith 2% (w/w) trypsin (TPCK-treated, Sigma) for 2 hours at 37° C. andthen with a further 2% (w/w) trypsin for 21 hours. One lot of nativepurified phosphatase in 100 mM Tris-HCl pH 8.0 was treated with 2% (w/w)trypsin for 20 hours at 37° C. and then with a further 2% (w/w) trypsinfor 6 hours. The peptides were purified as described below.

Another lot of purified native phosphatase was alkylated using 4-vinylpyridine as follows: To lyophilized phosphatase (75 μg) was added 40 μl0.5M Tris-HCl pH 7.5 containing 6M guanidium hydrochloride, 2 mM EDTAand 34 mM DTT. After addition of 1 μl 4-vinyl pyridine (Sigma), thereaction mixture was kept at room temperature (22° C.) for 1 hour. Thereaction was stopped by addition of 10 μl 1.4M DTT. Alkylatedphosphatase was then purified on HPLC with a C-1 reverse-phase column(TSK TMS 250; 0.46×4 cm) using a 20% to 70% ACN/0.06% TFA gradient (80%to 30% 0.1% TFA) in 30 minutes. The fractions absorbing at 218 nm werepooled and evaporated in a Speed-Vac vacuum centrifuge. The dried samplewas resuspended in 60 μl 70 mM Tris-HCl pH 9.1 and digested with 2%(w/w) lysylendopeptidase C (Wako Chemicals) for 2 hours at 37° C. Afteraddition of a further 2% (w/w) lysylendopeptidase C, the incubation at37° C. was prolonged to 26 hours. The peptides were purified asdescribed below.

Peptide purification and amino terminal sequencing

The peptides obtained by digestions were separated by HPLC on a C-18reverse-phase column (Vydac 218 TP B5; 0.46×25 cm) with a 90 minutegradient from 0 to 60% ACN/0.06% TFA (100 to 40% of 0.1% TFA).Absorbance at 218 nm was used for detection of peptides.

Amino terminal sequencing of the purified peptides, as well as thenative proteins, was done by degrading them in a gas-pulsed-liquid-phasesequencer (Kalkkinen and Tilgmann, 1988) J. Prot. Chem. 7:242-243). Thereleased PTH-amino acids were analyzed on-line by using narrow-borereverse-phase HPLC.

Carboxy terminal sequencing of phytase

One lot of purified phytase (53 μg) was digested with carboxypeptidase Y(Sigma, 0.6U) in 50 mM sodium acetate pH 5.6 containing 10% urea and0.05% SDS at room temperature (22° C.). Samples of the digestion werewithdrawn at various time points. These were dried in a Speed-Vac vacuumcentrifuge and derivatized with phenylisothiocyanate (PITC) according tothe amino acid analyzing kit Pico-Tag (Waters association). Analysis ofthe derivatized amino acids was performed by reverse-phase HPLC with thePico-Tag C-18 column, and quantified by identically derivatized aminoacid standards.

Results and Discussion

Sequences could be extracted from the peptides showing "doublesequences" (for both phytase and phosphatase, Table 4) since they werequantitatively different and/or the other sequence was already knownfrom peptides sequenced. Native phosphatase seemed to be somewhatresistant to lysylendopeptidase C digestion. After alkylation however,peptides of phosphatase were nicely obtained with lysyl-endopeptidase C.

The amino terminal sequence obtained from phytase (Nphy, #1081, SEQ IDNo. :50:!) was similar, but not identical, to the amino terminalsequence of A. ficuum phytase (LAVPASRNQSSGDT) SEQ ID No.:17:! reportedby Ullah, A. H. J. Prep. Biochem. 18:459-471 (1988). Peptides resultingfrom trypsin digestions are shown in Table 3. One peptide (10 phy) SEQID NO.:23:! had identical sequences with the internal peptide of A.ficuum phytase (MMQCQAEQEPLVRVLVNDR); SEQ ID No. :67: Ullah, A. H. J.Prep. Biochem. 18:459-471 (1988). Carboxyterminal sequencing of phytasegave the sequence XSA-OH.

No results were obtained from amino terminal sequencing of native andalkylated phosphatase (Table 4). One peptide (7Lpho) #817, SEQ ID No.:53: ! was, however, identical with the amino terminal sequence from A.ficuum acid phosphatase (pH optimum 2.5; FSYGAAIPQSTQEKQFSQEFRDG)published by Ullah, A. H. J. and Cummings, B. J., Prep. Biochem.17:397-422 (1987) and another (10 Lpho #941 SEQ ID No. :54:!) seems tobe a continuation of this. Peptide 3Tpho (peptide #1106 in Table 4; SEQID No. :61:) could also be a continuation of peptide 11Lpho (peptide#943-2 in Table 4; SEQ ID No. :60:) since it has four overlapping aminoacids: FSSG.

Peptide 1Lpho (peptide #816 in Table 4; SEQ ID No. :57:) contains theactive site consensus sequence RHGXRXP SEQ ID No. :18:! of phytases andphosphatases proposed by Ullah, A. H. J. et al., Biochem. Biophys. Res.Comun. 178:45-53 (1991). The peptide was highly homologous, but notidentical. One peptide of phytase (#675; SEQ ID No. :37:!, LKDPR) againcontained part of the KDPRA SEQ ID No. :19:! homologic sequence betweenA. ficuum phytase and different phosphatases reported by Ullah, A. H. J.et al., Biochem. Biophys. Res. Comun. 178:45-53 (1991).

The results indicate that A. niger phytase is homologous to A. ficuumphytase, but not identical. The same conclusion is reached in the caseof acid phosphatase (pH optimum 2.5).

                                      TABLE 3                                     __________________________________________________________________________    Amino acid sequence of isolated peptides of phytase                           Amino acid sequences of phytase peptides obtained as indicated in the         text. In                                                                      the case of uncertainty of the sequence, amino acids are shown in             brackets. X, stands for undetected                                            amino acids. The peptides are numbered (Xphy) according to appearance         (retention times) in the HPLC runs.                                           Phytase peptides (phy) obtained by trypsin digestion.                                   Peptide No.                                                                           Amino Acid Sequence.sup.a  Amino Acid Sequence Deduced                        from DNA                                                     SEQ ID No.: :!                                                                         (name)  sequence!.sup.b                                             __________________________________________________________________________     :20:!    132     Tyr--Tyr--Gly--His(Leu)--Gly--Ala--Gly--Asn--Pro--Leu--G                      ly--Pro--Thr--Gln                                                     (12 phy)                                                                               Tyr--Tyr--Gly--His.sub.-- Gly--Ala--Gly--Asn--Pro--Leu-                      -Gly--Pro--Thr--Gln!                                         :21:!    133     Thr--Gly--Tyr--Val--Gln(Asn)--Tyr--Val--Gln--Met--(Gln)                        not found in                                                                 DNA!                                                         :22:!    242     Ala--Gln--Pro--Gly--Gln--Ala--Ala--Pro--Lys                                    Ala--Gln--Pro--Gly--Gln--Ser--Ser--                                  (1 phy) Pro--Lys!                                                    :23:!    420     Leu--Tyr--Val--Glu--Met--Met--Gln--(Asn)--Gln--Ala--(Glu                      )--Gln--(Thr)--Pro--Leu--Val                                          (10 phy)                                                             :24:!             Leu--Tyr--Val--Glu--Met--Met--Gln--Cys--Gln--Ala--Glu--                      Gln--Glu--Pro--Leu--Val!                                     :25:!    410     Phe--Ile--Glu--Gly--Phe--Gln--Ser--Asp--Lys                           (13 phy)                                                             :26:!             Phe--Ile--Glu--Gly--Phe--Gln--Ser--Asp--Lys!                :27:!    416     Tyr--Ala--Phe--Leu--Lys  Tyr--Ala--Phe--Leu--Lys!                     (7 phy)                                                              :28:!    659     Gly--Leu--Ser--Phe--Ala--Arg  Gly--Leu--Ser--Phe--Ala--A                      rg!                                                                   (6 phy)                                                              :29:!    670 and 796                                                                           Val--Ile--Ala--Ser--Gly--Glu--Lys  Val--Ile--Ala--Ser--G                      ly--Glu--Lys!                                                         (2 phy)                                                              :30:!    418     Phe--Tyr--Gln--Arg  Phe--Tyr--Gln--Arg!                               (3 phy)                                                              :31:!    785     Phe--Tyr--Gln--Arg  = #418, 3phy, above! and                                  Asp--Ser--Phe--Val--Arg                                               (not pure)                                                                    (11 phy)                                                             :32:!             Asp--Ser--Phe--Val--Arg!                                    :33:!    248     Val--Leu--Val--Asn--Asp  not possible to compare to                           DNA!                                                                  (not pure)                                                           :34:!            Tyr Glu Ser Leu Gln                                          :35:!    784     Tyr--Glu--Ser--Leu--Thr--Arg  Tyr--Glu--Ser--Leu--Thr--A                      rg!                                                                   (9 phy)                                                              :36:!    675     Ser--Ala--Ala--Ser--Leu--Asn--Ser (a fragment of the                          trypsin enzyme)                                                       (not pure)                                                           :37:!            Leu--Lys--Asp--Pro--Arg  Leu--Lys--Asp--Pro--Arg!            :38:!    783     Val--Ile--Ala--Ser--Gly--Glu--Lys  small amount = #670                        and 796, above!                                                       (not pure)                                                           :39:!    (4 phy) Tyr--Pro--Thr--Glu--Ser--Lys  Tyr--Pro--Thr--Glu--Ser--L                      ys!                                                          :40:!    244     Tyr Phe Asn X Gly  not possible to compare to DNA!                    (not pure)                                                                            Asp Pro Ala X                                                :41:!    793     Leu--Glu--Asn/Pro--Asp/Phe--Leu--Asp/Ser--Gly/Leu--Phe/V                      al--Thr--Leu--)                                              :42:!             Leu--Glu--Asn--Asp--Leu--Ser--Gly--Val--Thr--Leu--Thr!      :43:!    792     Tyr--Tyr--Gly--His--Gly--Ala--Gly--Asn--Pro--Leu--Gly--P                      ro--Thr--Gln--Gly--Val--                                              (double Gly/Tyr--Ala--Asn--Glu--                                              sequence)                                                            :44:!    (15 phy)                                                                              Leu--Ile--Ala (= #132 (half of above), and                                    From this double sequence, the following sequences can                        be deduced                                                   :45:!            Val--Thr--Phe--Ala--Gln--Val--Leu--Ser                                         Val--Thr--Phe--Ala--Gln--Val--Leu--Ser!                                      and Tyr--Tyr--Gly--His--Gly--Ala--Gly--Asn--Pro--Leu--Gl                      y--Pro--Thr--Gln--Gly--                                                       Val--Gly                                                                       Tyr--Tyr--Gly--His--Gly--Ala--Gly--Asn--Pro--Leu--Gly--                      Pro--Thr--Gln--Gly--Val--                                                     Gly!                                                                          Tyr--Ala--Asn--Glu--Leu--Ile--Ala  Tyr--Ala--Asn--Glu--L                      eu--Ile--Ala!                                                :46:!    800     Phe--Ile--Glu--Gly--Phe--Gln--Ser--Thr                                         Phe--Ile--Glu--Gly--Phe--Gln--Ser--Thr!                              (13 phy)                                                             :47:!    797     Asp/Asn--Tyr--Leu--Gln--Ser--Leu--Lys                                          Asp--Tyr--Leu--Gln--Ser--Leu--Lys!                                   (13 phy)                                                             :48:!    795     (Odd behavior in peptide sequencing) Asn--Ile--Glu--Pro-                      -Phe--Gln--Val--                                                              Asn                                                                            not found in DNA sequence.!                                 :49:!    799     Val--Leu--Val--Asn--Asp--Arg {= #248,                                         above}  Val--Leu--Val--Asn--Asp--                                     (8 phy) Arg!                                                         :50:!    1081    Leu--Ala--Val--Pro--Ala--Ser--(Arg)--Asp--Gln--Ser--Thr-                      -X--Asp--Thr                                                          (Nphy)                                                                                 Leu--Ala--Val--Pro--Ala--Ser--Arg--Asn--Gln--Ser--Thr--                      Cys--Asp--Thr!                                               :51:!    C-terminal                                                                            --(Arg)--Ser--Ala--OH  Cys--Ser--Ala--End!                            (Cphy)                                                              __________________________________________________________________________     .sup.a peptide sequence X = amino acid not determined; slash (/) = either     one or the other of the two indicated amino acids may be present, the         assay was not definitive, () = the presence of the amino acids in             parenthesis is subject to question because of a weak signal of the PTH        amino acid;                                                                   .sup.b   ! =  peptide sequence deduced from DNA sequence, and #: peptide      number for identification                                                

                  TABLE 4                                                         ______________________________________                                        pH 2.5 acid phosphatase peptide sequences generated by either trypsin         (T) or endoproteinase Lys--Cys digestion of purified enzyme.                  Corresponding nucleotide positions are also listed                            Peptide Number                                                                             Peptide Sequence                                                                            Nucleotide Position                                ______________________________________                                        N-terminal #817;74pho                                                                      FSYGAAIPQSTQEK                                                                              193 . . . 234                                       SEQ ID No.:53:!                                                              #941;10/0pho QFSQEFRDGY    235 . . . 264                                       SEQ ID No.:54:!                                                              #938         YGGNGPY       280 . . . 300                                       SEQ ID No.:55:!                                                              #1111        VSYGIA        310 . . . 327                                       SEQ ID No.:56:!                                                              #816;1Lpho   RHGERYPSPSAGK 376 . . . 414                                       SEQ ID No.:57:!                                                              #847         DIEEALAK      415 . . . 438                                       SEQ ID No.:58:!                                                              #943-1;11 Lpho                                                                             ARYGHLWNGET   595 . . . 627                                       SEQ ID No.:59:!                                                              #943-2;11bpho                                                                              VVPFFSSG      628 . . . 651                                       SEQ ID No.:60:!                                                              #1106;3Tpho  FSSGYGR       640 . . . 660                                       SEQ ID No.:61:!                                                              #1110-1      QLPQFK        826 . . . 843                                       SEQ ID No.:62:!                                                              #1108        VAFGNPY       1384 . . . 1404                                     SEQ ID No.:63:!                                                              ______________________________________                                    

Example 5

The Cloning and Sequencing of the pH 2.5 Optimum Acid Phosphatase Genefrom Aspergillus niger

I. SUMMARY

The gene for pH 2.5 optimum acid phosphatase has been cloned andsequenced from Aspergillus niger. Translated nucleotide sequence yieldeda polypeptide of 479 amino acids for the pH 2.5 acid phosphatase. Thegene for this protein was isolated using oligonucleotide probes based onthe peptide sequence of the purified protein.

II. EXPERIMENTAL AND DISCUSSION

A. Design of oligonucleotide probes

Isolation of the gene encoding pH 2.5 acid phosphatase (AP) was madethrough hybridization of degenerate oligonucleotides designed frompeptide sequences. Several internal peptide fragments had been isolatedpreviously and sequenced from purified pH 2.5 AP from A. niger var.awamori strain ALKO 243 (ATCC 38854) as described earlier in thispatent.

A 17mer degenerate oligonucleotide, PHY-31, was designed from acidphosphatase peptide #816(1Lpho in Table 4 SEQ ID No. :57:!). Through theincorporation of a neutral inosine, one perfect match out of 64 possiblecombinations exists in PHY-31. The nucleotide sequence ofoligonucleotide PHY-31 and corresponding peptide sequence is shown inFIG. 1.

B. Hybridization specificity of the oligonucleotide probes

In order to evaluate the specificity of the degenerate oligonucleotides,they were end labelled with γ-³² P!-ATP to a high specific activityusing E. coli polynucleotide T4 kinase (BRL) and used to probe totalgenomic DNA from ALKO 243. Genomic DNA was isolated by a neutral lysismethod. Briefly, finely ground frozen dried mycelia was lysed with a 4%SDS-TE buffer. Cell debris was removed and supernatant was removed andextracted twice with an equal volumn of Tris-saturated phenol:chloroform(1:1). Genomic DNA was precipitated with NH₄ OAC and EtOH. Pelleted DNAwas purified by ultracentrifugation through CsCl and recovered asdescribed by Maniatis et al. Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA (1982)).Hybridization to genomic DNA with γ³² P! ATP labelled degenerateoligonucleotides (Maniatis et al. Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA(1982)) was done at 42° C. overnight on filters in oligonucleotidehybridization solution (6× SSPE, 0.5% SDS, 3× Denhardts, 100 μg/mltRNA). Non-specific binding was removed by washing the filters twice for30 minutes at room temperature with 2XSSC, 0.1% SDS, and once for 5minutes at 42° C. in fresh solution. Overnight exposure on Kodak X-OmatAR film with intensifying screens revealed positively hybridizing bands.

A. niger ALKO 243 genomic DNA was probed with pH 2.5 oligonucleotidePHY-31. Hybridization was performed as described above. Among theoligonucleotides for pH 2.5 AP, only PHY-31 gave relatively specifichybridization to genomic DNA. Hybridization to one predominant and oneminor band indicated sufficient specificity to use for screening thelibraries.

C. Isolation and characterization of the pH 2.5 Acid Phosphatase gene

Genomic DNA was partially digested with Sau3A in order to producefragments 10-23 kb in length. Digested DNA was ligated to BamHI-cutdephosphorylated Lambda Dash II vector arms (Stratagene). The ligatedDNA was packaged in vitro using Gigapack Gold packaging extracts(Stratagene). Packaged phage was used to infect E. coli strain P2392.The lambda library was screened with oligonucleotide PHY-31 for the pH2.5 AP gene under the conditions established with genomic hybridizationsin section (B). Twelve hybridizing plaques were picked for furthercharacterization. Bacteriophage DNA isolated from each of the candidateswas digested with restriction endonucleases and probed with eitherPHY-31 or a mixture of PHY-34 and PHY-35 which were derived from anindependent pH 2.5 AP peptide (FIG. 1). One of the clones, AP99,contained a 2.1 kb SphI fragment previously identified in genomicSouthern analysis, that hybridized strongly to both probes. Stronghybridization to two oligonucleotides derived from different peptidesequences suggested that AP99 contained pH 2.5 AP coding sequences. This2.1 kb SphI fragment was therefore subcloned into M13mp18 and M13mp19for sequencing. Translation of the nucleotide sequence of this subclonerevealed the peptide sequences including the N-terminal peptide (Table4). Immediately upstream of the N-terminal peptide is a typical fungalsecretion signal sequence beginning with a methionine initiation codonat position 136. All of the peptide sequences were present in a singleORF except #1108 (Table 4 SEQ ID No. :63! which begins at nucleotideposition 1384. Termination codons were identified in all three readingframes between nucleotides 1151 and 1384. These results necessitated theinclusion of an intron(s) in the 3' portion of the gene.

Identification of intron boundaries was made through the isolation andsequencing of pH 2.5 AP cDNA. The 3' region of pH 2.5 AP gene wasisolated from the corresponding cDNA by PCR amplification using pH 2.5AP specific primers. A. niger var. awamori ALKO 243 was grown in RNAbroth media consisting per liter of 2.0% corn starch (Sigma), 1.0%protease peptone (Difco), 30.0 g glucose, 5.0 g NH₄ NO₃, 0.5 g MgSO₄.7H₂O, 0.5 g KCl, 0.183 g FeSO₄.7H₂ O. Total RNA was isolated essentially bythe LiCl precipitation method of McAda and Douglas (McAda, P. C. et al.,Meth. Enzymol. 97:337-344 (1983)). Polyadenylated messenger RNA wasaffinity purified from total RNA by the use ofoligonucleotide(dT)-cellulose columns (Pharmacia) as specified by themanufacturer. Oligonucleotide PCR primers UPPHOS(5'GAATTCCGAGTCCGAGGTCATGGGCGCG-3') SEQ ID No. :67:! and DOWNPHOS(5'-GAATTCCCGGGACCTACCCCTCTGCAT-3') SEQ ID No. :16:! were synthesizedaccording to genomic sequences with flanking EcoRI restriction sites.UPPHOS and DOWNPHOS are inversely oriented and are separated by 978bases in the genomic clone. First strand synthesis was performed withthe BMB cDNA kit according to the manufacturer's recommendations with1.0 μg mRNA and DOWNPHOS. PCR amplifiction of the cDNA.mRNA complex witholigonucleotide primers UPPHOS and DOWNPHOS yielded a specific productof approximately 850 bps. PCR amplification of pAP-1 plasmid DNA withthe same primers yielded the expected product of 1006 bps. Gel purifiedcDNA PCR product was cut with EcoRI and subcloned into pUC-18 fordouble-stranded sequencing using the United States BiochemicalSequencase II kit. The primers amplified an 850 bp fragment from thecDNA and the expected 1006 bp fragment from cloned genomic DNA.Sequencing of the amplified cDNA fragment revealed the presence of threeshort introns, each exhibiting consensus fungal donor, lariat andacceptor sequences. The coding sequence is derived by splicing thenucleotides 136-916, 971-1088, 1141-1245, and 1305-1740. The resultingtranslated sequence codes for a protein of 479 aa as shown in FIGS.2(A-C).

The pH 2.5 AP polypeptide predicted from the nucleotide sequence has acalculated M_(r) of 52,678. The 2.1 kb SphI fragment in pUC18 (pAP-1)contained 135 bp of upstream pH 2.5 AP sequence.

Example 6

Aspergillus niger Phytase Production in Trichoderma reesei

II. EXPERIMENTAL PROTOCOLS

1. Bacterial strains, phage and plasmids

For subcloning and sequencing, the E. coil strains DH5α (Hanahan, D.,"Techniques for transformation of E. coli," in DNA Cloning, vol. 1,Glover, D. M., ed., IRL Press, Oxford, pp. 109-135 (1985); BethesdaResearch Laboratories, Gaithersburg, Md., USA) and XL-1-Blue (Bullock,W. O., et al., BioTechniques 5:376-378 (1987); Stratagene, La Jolla,Calif., USA) were used. E. coli Y1090 (r⁻) (Huynh, D. S., et al.,"Constructing and screening cDNA libraries in λgt1 and λgt11, " DNACloning, vol. 1, Glover, D. M., ed., IRL Press, Oxford, pp. 49-57(1985); Promega Biotec Protoclone GT System, Madison, Wis., USA) wasused as a host in λgt11 phage growing.

Aspergillus niger var. awamori ALKO 243 (ATCC 38854) was used as a donorof the phytase gene. T. reesei strains ATCC56765 (RutC-30), ALKO 233(VTT-D-791256, Bailey and Nevalainen, Enzyme Microb. Technol. 3:153-157(1981)) and ALKO 2221 were used as recipients for the phytase gene. ALKO2221 is a low aspartyl protease mutant derived from the strain ALKO 233by UV-mutagenesis.

The phage λgt11 (Promega) was used for making the gene library. Thephages were grown by the standard methods described by Silhavy et al.(Silhavy, T. J., et al., Experiments with Gene Fusions, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., USA (1984)).

As vectors for subcloning, pUC9 (Boehringer, Mannheim, FRG) and pALK307,a derivative of pIBI76 (IBI, New Haven, Conn., USA) were used. To obtainpALK307, an approximately 940 bp NaeI-Pvul fragment (actually 941 bp)has been deleted from pIBI76. This the only change in pIBI76. Theplasmid pAMH110 (Nevalainen, H., et al., "The molecular biology ofTrichoderma and its application to the expression of both homologous andheterologous genes," in Molecular Industrial Mycology, Leong and Berka,eds., Marcel Dekker Inc., New York, pp. 129-148 (1991)) contains theTrichoderma reesei cbh1 promoter and terminator areas. The plasmid p3SR2(Kelly and Hynes, EMBO J. 4:475-479 (1985)) contains the Aspergillusnidulans acetamidase gene. p3SR2 has been kindly donated by Dr. M. Hynes(University of Melbourne, Australia).

2. Growth media and culture conditions

E. coli cultivations were carried out at 37° C. overnight andcultivations of filamentous fungi at 30° C. for 5 to 7 days for enzymeproduction and for 2 days when mycelia was grown for DNA isolation.

E. coli were grown in L-broth (Maniatis, T., et al., Molecular Cloning,A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., USA (1982)) supplemented with ampicillin (50-100 μg/ml) whenneeded. PD agar slants (Potato Dextrose broth by Difco, Detroit, Mich.,USA) were used for growing the Aspergillus and the Trichoderma strains.Aspergillus niger ALKO 243 mycelium for DNA extraction was grown incomplete Aspergillus medium containing 2% (w/v) malt extract (Difco),0.1% (w/v) Bacto-peptone (Difco) and 2% (w/v) glucose. The plates andmedia for T. reesei transformations were as in Penttila et al.(Penttila, M., et al., Gene 61:155-164 (1987)). The transformants werepurified on selective acetamide-CsCl medium (Penttila, M., et al., Gene61:155-164 (1987)) before transferring to PD slants.

For plate screening of high phytase producers, T. reesei clonestransformed with the cbh1 promoter/phytase fusion were grown for 3 to 5days on Trichoderma minimal medium plates with no glucose andsupplemented with 1% sodium phytate (Sigma, St. Louis, Mo., USA), 1%Solka Floc and 1% proteose peptone (Difco). When the constructcontaining the phytase promoter was used for transformation, screeningof high phytase producers was carried out on plates containing 1% sodiumphytate and 1% proteose peptone but no sodium phosphate.

For phytase production A. niger ALKO 243 was grown for 5 days in a soyflour medium containing glucose and mineral salts (all from Merck,Darmstadt, FRG); the pH was adjusted to 5.0. For phytase expression fromthe cbh1 promoter, T. reesei transformants were grown for 7 days (250rpm) in a lactose based cultivation medium. For growing the T. reeseitransformed with the fragment containing the phytase promoter,Trichoderma minimal medium was supplemented with 50 g/l soy flour and nosodium phosphate was added.

The contents of the soy flower medium are as follows (per liter): 50 gof soy flower, 30 g glucose, 5.0 g NH₄ NO₃, 0.5 g MgSO₄, 7H₂ O, 0.5 gKCl, 0.183 g FeSO₄.7H₂ O, at pH 5.0. The lactose based cultivationmedium contains: 4% whey, 1.5% complex nitrogen source, 1.5% KH₂ PO₄,0.5% (NH₄)₂ SO₄, at pH 5.5.

3. DNA preparations

Plasmid DNA from E. coli (large scale) was isolated by using Qiagencolumns (Diagen GmbH, Dusseldorf, FRG) according to the manufacturer'sprotocol. For rapid screening the method of Holmes and Quigley (Anal.Biochem. 114:193-197 (1981)) was used. Chromosomal DNA from Aspergilluswas isolated from lyophilized mycelia as described in Clements andRoberts (Curr. Genet. 9:293-298 (1986)) and in Raeder and Broda (Lett.Appl. Microbiol. 1:17-20 (1985)).

4. Cloning procedures

The standard DNA methods described by Maniatis et al. (MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., USA (1982)) were mainly used. The restriction enzymes,T4-DNA-ligase, Klenow fragment of DNA polymerase I, T4 DNA polymerase,polynucleotide kinase and EcoRI methylase used in the DNA manipulationswere from Boehringer (Mannheim, FRG) and New England Biolabs (Beverly,Mass., USA). Mung bean nuclease was from BRL (Gaithersburg, Md., USA)and ExoIII from Pharmacia (Uppsala, Sweden). Each enzyme was usedaccording to the supplier's instructions.

For making the gene bank the chromosomal DNA was partially digested withHaeIII. EcoRI methylase treatment, size fractionation and packaging weredone as in Paloheimo et al. (Paloheimo, M., et al., Appl. Microbiol.Biotechnol. 36:584-591 (1992)). Fragments of a size of 2-8 kb were usedfor construction of the gene bank.

Subcloning into the plasmid vector was done by using standard DNAmethods (Maniatis, T., et al., Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA (1982)).DNA fragments for cloning or transformations were isolated fromlow-melting-point agarose gels (FMC Bioproducts, Rockland, Me., USA) bythe freeze-thaw-phenol method (Benson, S. A., Bio/Techniques 2:66-68(1984)) or by using the GeneClean® or Mermaid™ Kits (BIO 101 Inc., LaJolla, Calif., USA) according to the supplier's instructions.

Sequencing was carried out directly from the plasmids by the Sangermethod (Sanger, F., et al., Proc. Natl. Acad. Sci. USA 74:5463-5467(1977)) by means of SP6, 17, pUC/M13 primers and extension primers andthe Promega Sequenase sequencing kit (United States BiochemicalCorporation, Cleveland, Ohio, USA). Fusions between the cbh1 promoterand the phytase gene were sequenced by automated sequencer (AppliedBiosystems 373A, Foster City, Calif., USA) using Taq DyeDeoxy™Terminator Cycle Sequencing Kit (Applied Biosystems). Theoligonucleotides used were synthesized by an Applied Biosystems 381ASynthesizer except the pUC primers that were purchased from the UnitedStates Biochemical Corporation.

DNA probes were labeled by using the non-radioctive DIG-DNA Labellingand Detection Kit by Boehringer according to the supplier'sinstructions.

Hybridizations were done at 68° C. as in supplier's instructions(Boehringer). Amersham's Hybond N nylon filters were used in the plaquescreenings and in the Southern blot hybridizations.

When the plaques were screened with an antiserum, a phytase specificpolyclonal antiserum KH1236 was used. KH1236 was made against purifiedand deglycosylated phytase preparation (M. Turunen, Alko Ltd.) in theNational Public Health Institute (Helsinki, Finland). Anti-rabbit-IgGalkaline phosphate conjugate and color development substrates fromProtoBlot™ Immunoblotting system (Promega) were used to detect theimmunocomplexes.

5. Transformations

E. coli strains were transformed according to the method of Hanahan("Techniques for transformation of E. coli," in DNA Cloning, vol. 1,Glover, D. M., ed., IRL Press, Oxford, pp. 109-135 (1985)), and T.reesei strains as in Penttila et al. (Gene 61:155-164 (1987)). When theligated fragments were transformed (the D-transformation in Table 7) theligation mixture was not further purified but was used as such in thetransformations. Prior to sporulating on potato dextrose agar (PD)slants T. reesei transformants were transferred on the selective mediumand purified through conidia.

6. Enzyme and protein measurements

For the enzyme assays T. reesei mycelium was separated from the culturemedium by centrifuging for 15 to 30 min at 5,000 to 10,000 rpm (SorvallSS-34, Dupont Company, Wilmington, Del., USA). A. niger cultures werecentrifuged for 40 min at 10,000 rpm (Sorvall SS-34). The phytaseactivity was measured from the culture supernatant as the amount ofinorganic phosphate released by enzymatic hydrolysis of sodium phytatesubstrate at 37° C. as described earlier. One phytase normalized unit(PNU) is defined as the amount of phytase activity produced by the A.niger ALKO 243 strain under the cultivation conditions used.

The phytase production on the sodium phytate assay plates was visualizedby pouring the reagent C (3:1:1 ratio of 1M H₂ SO₄, 2.5% ammoniummolybdate, 10% ascorbic acid) on the plates and incubating them at 50°C. for 15 minutes. The reduction of the phosphomolybdate complex leadsto bluish color.

Amyloglucosidase activity (AGU) was measured by using 1% Zulkowskystarch (Merck) as a substrate and measuring the amount of the releasedglucose units by boiling with DNS reagent (see below) after 10 min ofreaction at 60° C. at pH 4.8. Proteases (HUT) were measured at pH 4.7 asin Food Chemicals Codex (1981) by using 2% haemoglobin (Sigma) as asubstrate. Endoglucanase (ECU) and cellobiohydrolase (FPU) activitieswere measured as in IUPAC's Measurement of Cellulase Activities (IUPACCommission on Biotechnology, Measurement of Cellulase Activities,Biochemical Engineering Research Centre, Indian Institute of Technology,Delhi, India, pp. 5-7 and 10-11 (1981)). 1% hydroxyethylcellulose (FlukaAG) in 50 mM sodium-citrate buffer (pH4.9) and Whatman no. 1 paper wereused as substrates, respectively. DNS used differed from that describedat the IUPAC's protocol and was made by first diluting 50.0 g2-hydroxy-3,5-dinitrobenzoic acid (Merck) into 4 l of deionized water.Then, 80.0 g NaOH was added slowly by using the magnetic stirrer and1,500 g sodium-potassium tartrate (Merck) was added and diluted byheating the solution (maximum temperature 45° C.). The total volume wasadjusted to 5 l, the solution was filtered through Whatman no. 1 and wasprotected from light.

CBHI protein was measured from the culture supernatant by running aSDS-polyacrylamide gel and detecting the CBHI protein band (T. reeseiALKO 233 and ALKO 2221 strains) or by dot blotting samples using theSchleicher & Schuell's (Dassel, FRG) Minifold™ Micro-Sample FiltrationManifold (T. reesei ATCC56765). CBHI protein was detected by using CBHIspecific monoclonal antibody CI-89 (Aho, S., et al., Eur. J. Biochem.200:643-649 (1991)) and anti-mouse-IgG alkaline phosphate conjugate(Promega). Visualization of the immunocomplexes was done as in theplaque screening.

7. SDS-PAGE and Western blot analysis

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)and Western blot analysis were done as in Laemmli (Laemmli, U. K.,Nature 227:680-685 (1970)) and as in Towbin et al. (Towbin, H., et al.,Proc. Natl. Acad. Sci. USA 76:4350-4354 (1979)). Visualization of thephytase protein in Western blots was done by using the polyclonal rabbitantiserum KH1236. Visualization of the immunocomplexes was done as inthe plaque screening.

8. Polymerase Chain Reaction (PCR)

The PCR reactions were performed by Techne Thermal Cycler PHC-2 (TechneLtd., Cambridge, UK) in 100 μl volumes. The reaction mixtures contained0.2 mM of each dNTP (3'-deoxynucloside-5'-triphosphate, Pharmacia) in 10mM Tris buffer (pH 8.3), 50 mM KCl, 1.5-2.0 mM MgCl₂ and 0.01% (w/v)gelatin. The protocol used was the following: 95° C. (plasmid) or 100°C. (chromosomal DNA)/5 min before adding the Taq DNA polymerase (1-2units, Cetus Corp., Emeryville, Calif., USA) and 100 μl overlay ofparaffin oil, denaturation 95° C./1 min, annealing 60° C./1 min (in theinverse PCR 52° C.), extension 72° C./2 min (in the inverse PCR 2.5 min)for 30 cycles. The final extension time was 9 min to ensure completionof the strand synthesis. When a plasmid or DNA fragment was used as atemplate, the amount of the template used was 5-10 ng and 20-50 pmol ofeach primer was added. When chromosomal DNA was used as a template, thecorresponding amounts used were 100 ng and 50-100 pmol. The circulartemplate for the inverse PCR was done from the digested chromosomal DNAas in Innis et al (Ochman, H., et al., "Amplification of flankingsequences by inverse PCR," in PCR Protocols, A Guide to Methods andApplications, Innis, M. A., et al., eds., Academic Press, San Diego, pp.219-227 (1990)). PCR fragments were purified by GeneClean® or Mermaid™Kit (from an agarose gel if needed) or by Qiagen tips. The ends of thefragments were filled by using the DNA polymerase I Klenow fragment.

II. RESULTS

A. Molecular cloning of the Aspergillus niger phytase

1. Production of the phytase probe by nested PCR amplification

The oligonucleotide primers used in the PCR reactions and thecorresponding amino acid sequences of the phytase ALKO 243 peptides #792(phy 15) and #420 (phy 10) (Table 3, as described earlier) are shown inFIG. 3 (nucleotides 1409-1480 and 1727-1762 in the phytase sequence, seeFIGS. 5(A-D). Two primary PCR reactions were done. In reaction A, senseoligonucleotide 1 (#792) and antisense oligonucleotide 4 (#420) wereused; in reaction B, sense oligonucleotide 3 (#420) and antisenseoligonucleotide 2 (#792) were used. Primary PCR reaction A gave a singleband of about 400 bps while PCR amplification from the reaction B gaveno product. Thus it was concluded that the region coding for the peptide#792 was located on the 5'-side in the phytase sequence compared to thatcoding for the peptide #420. The primary PCR fragment (A) was used as atemplate for the second PCR with internal primers from the peptidesequences: sense oligonucleotide 2 and antisense oligonucleotide 3. PCRamplification from the second PCR reaction gave a specific product ofabout 350 bps. The PCR fragment was cloned to SmaI digested pUC9 andsequenced. The amino acid sequence deduced from the DNA sequencecontained also the known amino acids from the peptide #792 and #420 thatwere not coded by the primer sequences. The approximately 350 bp PCRfragment was used to probe the DNA bank.

2. Screening of the DNA bank

The gene bank contained approximately 1.9×10⁶ pfu (plaque formingunits)/ml, of which approximately 99.5% had an insert. From about 80,000plaques that were screened, two positive clones Hae2-6 and Hae1-5 werefound. The clones were isolated, purified and the inserts (5.6 and 5.2kb) were subcloned to EcoRI cut pALK307. The clones also reacted withthe phytase antiserum KH1236. The inserts were restriction mapped andthe PCR fragment was found to hybridize to the about 1 kb BamHI-SphIrestriction fragment of the clones (see FIG. 4 for the hybridizationarea in the phytase sequence). The sequence of the clone Hae2-6 thatcontained more of the 5'-sequence coded for 15 internal tryptic peptidesequences but the N-terminal amino acid sequence was not found. TheN-terminal and the promoter area containing phage clones were screenedfrom the gene bank by using a 5'-probe made by inverse PCR (Ochman, H.,et al., "Amplification of flanking sequences by inverse PCR," in PCRProtocols, A Guide to Methods and Applications, Innis, M. A., et al.,eds., Academic Press, San Diego, pp. 219-227 (1990)).

3. Amplification of the 5'-end and the promoter sequence of the phytasegene by inverse PCR

Restriction enzyme digestions of the genomic DNA and Southernhybridizations with the 350 bp PCR probe showed that digestions of thegenomic DNA with SalI produced fragments of suitable size (1-3 kb) forcircularization and amplification with PCR. The primers used for inversePCR were from bases 1243-1257 (antisense primer) and 1304-1321 (senseprimer) areas of the phytase sequence (see FIGS. 5(A-D)). Inverse PCRwith SalI digested ALKO 243 DNA created a PCR band of about 1.2 kb. The350 bp PCR fragment hybridized to the inverse PCR fragment and bysequencing the subcloned fragment it was confirmed that it contained theupstream parts of the phytase gene and also the N-terminal peptidesequence was included.

4. Isolation of the complete phytase gene

The 1.2 kb PCR fragment obtained from the inverse PCR was used as aprobe to screen 80,000 plaques from which seven positive plaques couldbe identified. The complete phytase gene was isolated on an about 6 kbinsert of a phage clone.

The about 2.4 kb SphI fragment containing the phytase gene and thepromoter area was subcloned into pALK307 (cut with SphI) to give pALK169(FIG. 4). The restriction map of the phytase containing SphI fragmentand the location of the phytase gene in the fragment are shown in theplasmid map.

The phytase sequence is shown in FIGS. 5(A-D). The sequence coding forthe phytase protein corresponds to the phytase sequence of Aspergillusficuum published in the Gist brocades (Delft, Netherlands) PCT patentapplication (EP420,358 or WO91105053) with 12 differences in the deducedamino acids. Each difference in the deduced amino acid was due to onenucleotide's change and might be due to differences between the strains.Also, in the sequence coding for the structural gene, 33 nucleotidedifferences were found that did not lead to differences in the deducedamino acid sequence. In the signal sequence, there were differences intwo nucleotides (the other lead to a difference in the deduced aminoacid) and in the proposed intron area 8 differences could be found. Theoverall match per length (nucleotide sequences from the first ATG to theSTOP codon TAG) between the two sequences was 96.3%. The differencesfound between the two phytase sequences, Gistbrocades' and Alko's, areshown in the Table 5.

                  TABLE 5                                                         ______________________________________                                        Nucleotide and amino acid differences between Gist's and Alko's phytase       sequences.                                                                                                  Alko's       Alko's                             area   nt no   as no  Gist's nt(s)                                                                          nt(s) Gist's aa                                                                            aa                                 ______________________________________                                        signal 39      (-7)   CTG     CTA   Leu    Leu                                sequence                                                                             40      (-6)   TCT     GCT   Ser    Ala                                proposed                                                                             59      (-)    A       G     (-)    (-)                                intron 61      (-)    A       T     (-)    (-)                                       65      (-)    A       T     (-)    (-)                                       72      (-)    A       G     (-)    (-)                                       85      (-)    C       T     (-)    (-)                                       86      (-)    C       T     (-)    (-)                                       88      (-)    T       G     (-)    (-)                                       136     (-)    T       A     (-)    (-)                                structural                                                                           191     11     AGT     ACT   Ser    Thr                                gene   210     17     CAG     CAA   Gln    Gln                                       258     33     GCA     GCG   Ala    Ala                                       287     43     GTC     GCC   Val    Ala                                       300     47     GAG     GAT   Glu    Asp                                       312     51     GGA     GGT   Gly    Gly                                       369     70     GAC     GAG   Asp    Glu                                       419     87     GCG     GTG   Ala    Val                                       369     91     GAC     GAT   Asp    Asp                                       501     114    GAA     GAG   Glu    Glu                                       549     127    CGG     CGA   Arg    Arg                                       565     136    GTT     ATT   Val    Ile                                       570     137    CCA     CCG   Pro    Pro                                       613     152    AAG     GAG   Lys    Glu                                       624     155    ATC     ATT   Ile    Ile                                       669     170    CCC     CCG   Pro    Pro                                       756     199    TTC     TTT   Phe    Phe                                       809     217    GTC     GCC   Val    Ala                                       846     229    TCC     TCT   Ser    Ser                                       849     230    GGT     GGC   Gly    Gly                                       976     273    AAC     CAC   Asn    His                                       997     280    TTG     CTG   Leu    Leu                                structural                                                                           1005    282    AAG     AAA   Lys    Lys                                gene   1008    283    TAT     TAC   Tyr    Tyr                                       1020    287    GGT     GGC   Gly    Gly                                       1083    308    CTG     CTC   Leu    Leu                                       1113    318    AGT     AGC   Ser    Ser                                       1125    322    ACT     ACC   Thr    Thr                                       1136    326    AGC     AAC   Ser    Asn                                       1140    327    CCG     CCA   Pro    Pro                                       1149    330    TTT     TTC   Phe    Phe                                       1182    341    TCG     TCC   Ser    Ser                                       1185    342    CAT     CAC   His    His                                       1188    343    GAC     GAT   Asp    Asp                                       1203    348    TCC     TCT   Ser    Ser                                       1206    349    ATT     ATC   Ile    Ile                                       1218    353    TTA     TTG   Leu    Leu                                       1245    362    CTA     CTG   Leu    Leu                                       1284    375    GGA     GGG   Gly    Gly                                       1321    388    TTG     CTG   Leu    Leu                                       1344    395    TGT     TGC   Cys    Cys                                       1350    397    GCG     GCC   Ala    Ala                                       1413    418    CCG     CCA   Pro    Pro                                       1414    419    GTT     ATT   Val    Ile                                       1499    447    TTT     TCT   Phe    Ser                                ______________________________________                                         Nucleotide and amino acid differences between Gist's and Alko's phytase       sequences. The nucleotide numbers are counted from the firstMet (ATG) in      the signal sequence and amino acid numbers from the Nterminal Leu (see        FIG. 5 (AD). The amino acids, if different between the two sequences, are     written by bold letters.                                                 

B. Construction of the plasmids for overexpression of phytase inTrichoderma reesei

1. The PCR fragments for the precise cbh1-phytase fusions

The fusions between the cbh1 promoter and the phytase signal sequence,and between the cbh1 signal sequence and the phytase gene were done byPCR. The phytase signal sequence or the phytase protein N-terminalsequence start precisely where the corresponding cbh1 sequences wouldstart (for the cbh1 sequence, see Shoemaker, S., et al., Bio/Technology1:691-696 (1983)). The primers used for the PCR fragments are shown inFIG. 6. To construct pALK171 (phytase signal sequence), we made use ofthe SacII site in the cbh1 promoter area in the 5'-PCR primer. The XhoIsite (14 nucleotides from the N-terminal of the phytase gene) was usedin the 3'-primer. The 5'-primer was a 39-mer that contained a "tail" of19 nucleotides of the cbh1 promoter sequence (preceding the signalsequence) and 20 nucleotides from the phytase signal sequence. The3-primer was a 22-mer. In the construction of pALK172 (cbh1 signalsequence), we made use of the SfiI site in the cbh1 signal sequence inthe 5'-primer and SalI site of the phytase (762 nucleotides from theN-terminal) in the 3'-primer. In this case, the 5'-primer was a 46-mercontaining a "tail" of 28 nucleotides and 18 nucleotides of the phytaseN-terminal sequence; the 3'-primer was a 24-mer. In all the primers,three to five extra nucleotides were added to the ends of the PCRfragments after the restriction site sequences to ensure a correct cut.pALK169 was used as a template in the PCR reactions.

Fragments of the expected lengths were obtained from the PCR reactions:fragment containing the wanted fusion was 202 bps for pALK171 and 800bps for pALK172.

2. Construction of plasmids with the cbh1 promoter: pALK171 (phytasesignal sequence) and pALK172 (cbh1 signal sequence)

The plasmid pALK170L was made by cutting the phytase gene from pALK169as an SphI-XhoI fragment and ligating it to SphI-XhoI cut pALK307. The202 bp PCR fragment containing the cbh1 promoter and phytase signalsequence was cut with XhoI and ligated to pALK170L that had been cutwith XbaI, treated with the DNA polymerase I Klenow fragment and cutwith XhoI (pALK170X, FIGS. 7(A-B)). The fusion and the PCR fragmentareas were sequenced to ensure that no mistakes had occurred in the PCRamplification. Phytase fragment containing the fusion was isolated as anSphI (treated with the T4 DNA polymerase)-SacII fragment and wasinserted between the cbh1 promoter and terminator areas of the plasmidpAMH110 previously digested with NdeI (filled in with the DNA polymeraseI Klenow fragment) and SacII. The plasmid obtained (pALK171X, FIGS.7(A-B)) contains the phytase gene precisely fused to the cbhI promoter.To construct pALK171, the amdS gene (selectable marker) was isolatedfrom p3SR2 as a SphI-KpnI fragment, the ends were treated by the T4 DNApolymerase, and amdS was then ligated to the SphI site of pALK171X(treated with the T4 DNA polymerase). The approximately 7.5 kb linearfragment that contained no bacterial sequences was isolated from pALK171by cutting with XbaI and was used for the transformations.

pALK172 was constructed essentially the same way as pALK171 (see FIGS.8(A-B)). The 800 bp PCR fragment was cut with SalI and ligated to XbaI(filled in with the DNA polymerase I Klenow fragment), SalI cutpALK170L. Also in this case, the fusions and the sequence of the PCRfragment were checked by sequencing. Phytase-PCR fragment fusion wasisolated from pALK170S as an SphI (treated with the T4 DNApolymerase)-SfiI fragment and ligated to pAMH110 that had been cut withNdeI (filled in with the DNA polymerase I Klenow fragment)-SfiI. Toconstruct pALK172, the fragment containing the amdS gene was ligated topALK172S in the same way as when constructing pALK171. XbaI was usedalso in this case to cut out from the vector backbone the linearfragment that was used in the transformations.

3. Construction of the plasmids with the phytase promoter: pALK173A andpALK173B

The phytase gene with its own promoter was isolated as an SphI fragmentfrom pALK169 and ligated into the KpnI site of p3SR2 (in both cases theends of the fragments were filled in by the T4-polymerase) resulting inabout 11.2 kb plasmids pALK173A and pALK173B (see FIG. 4 for pALK169 andFIGS. 7 and 8 for p3SR2 map). In pALK173A the two genes, phytase andamdS are in a parallel orientation, in pALK173B, they are in oppositeorientations (FIG. 9). EcoRI was used to linearize pALK173A and pALK173Bwhen linearized plasmids were used for transformations.

C. Transformation of Trichoderma reesei and screening of thetransformants

T. reesei ATCC56765, ALKO 233 and ALKO 2221 strains were transformedwith circular plasmids and with the XbaI fragments from the plasmidspALK171 and pALK172 (cbh1 promoter). T. reesei ALKO 233 and 2221 strainswere also transformed with the linearized pALK171 and pALK172 plasmidsas well as with circular and linear pALK173A and pALK173B (phytasepromoter) plasmids. Transformation frequencies (transformants/μg of DNA)varied from 3 to 60 when the fragments isolated from pALK171 or pALK172were used. When pALK171 or pALK172 circular plasmids were used intransformations, the frequencies were about 50/μg for T. reesei ALKO 233and ALKO 2221 and about 100/μg for T. reesei ATCC56765. Transformationfrequencies obtained when linearized plasmids were used were about100/μg. When pALK173A or pALK173B were used in transformations thefrequencies were from 6 to 26 for the linear plasmid and from 6 to 20for the circular plasmid.

Regeneration frequency of the sphaeroplasts varied from 4.5% to 13.2%for T. reesei ALKO 233 and ALKO 2221 strains and was 1-2% for the T.reesei ATCC56765 strain.

The amount of the transformants that were screened for the phytaseproduction on plates are shown in Table 6. Those clones that clearlyproduced a blue colored halo around the colony were counted as positiveclones.

                  TABLE 6                                                         ______________________________________                                        Plate assay positive transformants and total number of tested clones          Plasmid    ALKO 233   ALKO 2221  ATCC56765                                    ______________________________________                                        pALK171                                                                       fragment   49% (47/96)                                                                              46% (33/71)                                                                              23% (15/66)                                  circular plasmid                                                                         35% (6/17) 23% (5/22) 32% (22/68)                                  linear plasmid                                                                           41% (29/71)                                                                              49% (27/55)                                                                              ND                                           pALK172                                                                       fragment   47% (48/103)                                                                             30% (34/113)                                                                             11% (8/72)                                   circular plasmid                                                                         17% (4/23) 13% (2/15) 12% (12/104)                                 linear plasmid                                                                           37% (22/59)                                                                              24% (11/45)                                                                              ND                                           pALK173A                                                                      circular plasmid                                                                         75% (9/12) 70% (14/20)                                                                              ND                                           linear plasmid                                                                           67% (10/15)                                                                              64% (14/22)                                                                              ND                                           pALK173B                                                                      circular plasmid                                                                         40% (4/10) 63% (15/24)                                                                              ND                                           linear plasmid                                                                           63% (10/16)                                                                              67% (8/12) ND                                           ______________________________________                                         Table 6. Plate assay positive transformants and total number of tested        clones. The number of the plate assay positive transformants and the tota     number of phytase plate assay tested transformants are shown. Only those      transformant strains that grew well both on the selection slant and on th     plate assay are included. As positive phytase producers are counted those     strains that clearly showed phytase activity on the plate assay.         

The transformants that seemed to be the best producers on the plateassay were grown on shake flasks. Inocula were taken either directlyfrom acetamide slants or from PD slants after purification throughconidia. Of the T. reesei ATCCS6765, ALKO 233 and ALKO 2221, transformedwith the fragments from pALK171 and pALK172, from 7 to 16 clones fromeach set of transformants were purified and the phytase production wastested in shake flask cultivations. When circular plasmids pALK171 orpALK172 had been used in transformations, 13 and 12 purified T. reeseiATCC56765, and from four to seven T. reesei ALKO 233 and ALKO 2221transformant strains were grown, respectively. When linearized pALK171or pALK172 plasmids were used, about 20 transformant strains from eachwere grown in shake flasks. Of the T. reesei ALKO 233 clones transformedwith the linear pALK173A/B plasmids, seven PALK173A and two pALK173B(and with the circular plasmids four pALK173A and three pALK173B)transformants indicating phytase activity on plates were purified andtested in shake flask cultivations. For the T. reesei ALKO 2221transformants the corresponding amounts tested in shake flaskcultivations were as follows: for the linear plasmids, three pALK173Aand five pALK173B and for the circular plasmids six pALK173A and sixpALK173B transformants.

D. Phytase production by the Trichoderma transformants

The best phytase production levels from transformants of T. reeseiATCC56765, ALKO 233 and ALKO 2221, without E. coli sequences, when thecbh1 promoter (pALK171 and pALK172 fragments) was used are shown inTable 7.

                                      TABLE 7                                     __________________________________________________________________________    Phytase production and enzyme profiles of the best T. reesei phytase          producing                                                                     transformants with no E. coli sequences                                       Strain Fragment                                                                           Transformant                                                                         PNU/ml                                                                            CBHI                                                                             AGU/ml                                                                             HUT/ml                                                                            ECU/ml                                                                            FPU/ml                                 __________________________________________________________________________    ALKO 233                                                                             none        <   (+)                                                                              79   170 690 3.2                                           pALK171                                                                            E16    3,590                                                                             (-)                                                                              ND   ND  ND  ND                                                 A53    2,820                                                                             (+)                                                                              91   140 385 2.1                                                D1     2,740                                                                             (+)                                                                              ND   ND  ND  ND                                                 A52    2,580                                                                             (+)                                                                              88   190 505 3.0                                                A13    2,570                                                                             (+)                                                                              82   215 905 1.1                                           pALK172                                                                            E101   2,000                                                                             (+)                                                                              ND   ND  ND  ND                                                 A12    1,900                                                                             (+)                                                                              104  ND  <1,000                                                                            ND                                                 D4     1,570                                                                             (+)                                                                              ND   ND  ND  ND                                                 E70    1,460                                                                             (+)                                                                              ND   ND  ND  ND                                                 E80    1,430                                                                             (-)                                                                              ND   ND  ND  ND                                     ALKO 2221                                                                            none        <   (+)                                                                              <10  <15 740 3.5                                           pALK171                                                                            D2     3,200                                                                             (+)                                                                              32   42  460 2.4                                                A9     2,840                                                                             (+)                                                                              <10  <20 480 2.6                                                A24    2,760                                                                             (+)                                                                              <10  <20 340 1.5                                                E3     2,670                                                                             (-)                                                                              ND   ND  ND  ND                                                 D1     2,390                                                                             (+)                                                                              <10  32  410 2.5                                           pALK172                                                                            B17    3,480                                                                             (+)                                                                              ND   ND  ND  ND                                                 E6     2,860                                                                             (+)                                                                              ND   ND  ND  ND                                                 D4     2,590                                                                             (+)                                                                              27   29  360 1.4                                                E8     2,480                                                                             (+)                                                                              ND   ND  ND  ND                                                 A96    2,380                                                                             (+)                                                                              <16  31  430 2.6                                    ATCC56765                                                                            none        <   (+)                                                                              1.1  57  200 0.8                                           pALK171                                                                            B1     1,620                                                                             (+)                                                                              <1.0 55  145 <1.0                                               A11    1,300                                                                             (+)                                                                              <1.0 58  145 <1.0                                               A21    1,280                                                                             (+)                                                                              <1.0 ND  120 ND                                                 B25    1,090                                                                             (+)                                                                              <1.0 ND  60  ND                                            pALK172                                                                            B19    1,780                                                                             (+)                                                                              <1.0 52  120 <1.0                                               B1     1,180                                                                             (+)                                                                              <1.0 53  120 <1.0                                               B11    1,080                                                                             (+)                                                                              <1.0 ND  100 ND                                     A. niger ALKO                                                                        none        1   ND 46   31  35  0.0                                    243                                                                           __________________________________________________________________________     Phytase production and enzyme profiles of the best T. reesei phytase          producing transformants with no E. coli sequences. Phytase activities as      PNU/ml, background activities (AGU, HUT, ECU and FPU/ml) and the              production of CBHI protein (+/-) in the supernatant are shown. T. reesei      ATCC56765, ALKO 233 and ALKO 2221 strains were transformed with the XbaI      fragment from the plasmid pALK171 or pALK172 (cbhl promoter, no E. coli       sequences). Strains were purified through conidia before cultivations. Th     values shown are averages from two shake flask cultivations. A "less than     sign (<) means that the value was below the detection level. ND = not         determined.                                                              

About 3600 PNU/ml was obtained with the best transformant. About thesame level of production could be achieved by using both the strains T.reesei ALKO 233 and ALKO 2221. The best phytase producing T. reeseiATCC56765 transformant produced about 1,800 PNU/ml. Both the phytase andthe cbh1 signal sequence seemed to work equally well and the same levelsin phytase production could be achieved when using T. reesei ALKO 2221or ATCC56765 as a host strain. In T. reesei ALKO 233 the level ofphytase activity produced was higher when the phytase signal sequencewas used.

Some of the transformants did not produce any detectable CBHI proteinwhich most probably indicates integration of the transforming DNA to thecbh1 locus. The absence of the CBHI protein did not affect theproduction levels in the screened transformants, i.e., good producerswere found both among the transformants producing normal amounts of CBHIas well as among CBHI negative strains.

The best phytase production levels obtained by the use of pALK171 andpALK172 circular and linear plasmid are shown in the Table 8. The bestproduction yields were obtained with the T. reesei ALKO 2221 that hadbeen transformed with the linear plasmid pALK171 (phytase signalsequence).

                  TABLE 8                                                         ______________________________________                                        Phytase production by the T. reesei strains transformed with                  circular or linear plasmid pALK171 or pALK172                                 Strain  Plasmid   Plasmid form                                                                             Transformant                                                                           PNU/ml                                  ______________________________________                                        ALKO 233                                                                              pALK171   circular   C13        650                                                                C23        530                                                                C4         240                                                     linear     A22      1,610                                                                A21      1,270                                                                A17      1,180                                           pALK172   circular   C13      1,360                                                                C21        540                                                                C1         500                                                     linear     A20      1,420                                                                A27      1,330                                                                A32        980                                   ALKO 2221                                                                             pALK171   circular   C2       1,630                                                                C8       1,290                                                                C32        810                                                     linear     A14      3,800                                                                A8       3,660                                                                B14      3,610                                           pALK172   circular   C3         480                                                                C4         190                                                                C6         170                                                     linear     B18      2,060                                                                A36      1,790                                                                B9       1,390                                   ATCC56765                                                                             pALK171   circular   A74      2,030                                                                A75      1,980                                                                B11      1,870                                           pALK172   circular   B3       2,250                                                                B23      1,970                                                                B1       1,030                                   ______________________________________                                         Table 8. Phytase production by the T. reesei strains transformed with         circular or linear plasmids pALK171 and pALK172. Phytase activities as        PNU/ml in the culture supernatants of the three best phytase producing        transformants of each type are shown. T. reesei ATCC56765 transformants       were purified through conidia before cultivations and the results are         averages from two shake flask cultivations. Inocula for cultivations of       the T. reesei ALKO 233 and ALKO 2221 transformants were taken from the        acetamide slants and the results shown are from one shake flask               cultivation.                                                             

Also the A. niger phytase promoter can promote the expression of thegene in Trichoderma. However, the enzyme yields obtained are much lowerthan with the cbh1 promoter homologous to T. reesei: the activitiesobtained from the culture supernatants of transformants containing thephytase's own promoter were from about 1 to about 14 PNU/ml for the T.reesei ALKO 233 transformants and from about 6 to about 120 PNU/ml forthe T. reesei ALKO 2221 transformants (Table 9).

                  TABLE 9                                                         ______________________________________                                        Phytase production by the T. reesei strains transformed with                  circular or linear plasmid pALK173A or pALK173B                               Strain  Plasmid   Plasmid form                                                                             Transformant                                                                           PNU/ml                                  ______________________________________                                        ALKO 233                                                                              pALK173A  circular   C25      14.2                                                                 C29      6.6                                                                  C23      1.2                                                       linear     D4       8.8                                                                  D18      5.8                                                                  D6       3.9                                             pALK173B  circular   C8       3.5                                                                  C6       <                                                                    E10      <                                                         linear     D13      5.5                                                                  D31      <                                       ALKO 2221                                                                             pALK173A  circular   E3       32.5                                                                 E5       31.7                                                                 C22      25.0                                                      linear     D24      37.5                                                                 D36      8.3                                                                  D17      5.8                                             pALK173B  circular   C13      115.8                                                                C28      65.0                                                                 C27      50.8                                                      linear     D9       36.7                                                                 D26      22.5                                                                 D21      18.3                                    ______________________________________                                         Table 9. Phytase production by T. reesei transformants transformed with       circular or linear plasmid pALK173A or pALK173B (phytase promoter).           Phytase activities as PNU/ml in the culture supernatants of the               transformants are shown. Transformants have been purified through conidia     before cultivation. The results shown are from one shake flask                cultivation. A "less than" sign (<) means that the value was below the        detection level.                                                         

E. The enzyme background in the phytase preparations produced by T.reesei

Phytase is expressed in the T. reesei strains in high amounts and thebackground of other enzyme activities in the supernatants of T. reeseitransformants is different from those in the Aspergillus supernatant(Table 7). Both endoglucanase and cellobiohydrolase activities aresubstantially higher when T. reesei is used as a production hostcompared to A. niger. The T. reesei strains used also producedproportionally less glucoamylase activity than the A. niger ALKO 243strain.

F. Phytase protein produced by the Trichoderma transformants

Samples from the growth media of the transformants (pALK171 fragment)and the nontransformed T. reesei strains ATCC56765, ALKO 233 and ALKO2221 were analyzed in Western blots (FIG. 10). Briefly, the followingsamples were analyzed: Lane 1: 50 ng of purified Aspergillus ALKO 243phytase; Lane 2: 15 ng of endoF-treated Aspergillus ALKO 243 phytase;Lanes 3 and 10: T. reesei ALKO 233; Lanes 4-5 and 11-12: T. reesei ALKO233 transformant 171FR/A4 and A13, respectively; Lanes 6 and 13: T.reesei ALKO 2221; Lanes 7-8 and 14-15: T. reesei ALKO 2221 transformant171FR/A5 and A9, respectively; Lane 9: T. reesei ALKO 2221 transformantD2; Lane 16: T. reesei ATCC56765; Lanes 17, 18, 19: T. reesei ATCC56765transformants 171FR/A21, A11, and A23, respectively. In each case, 2 μlof 1: 10 dilution of the culture supernatant were run on the gel. 171FRis the host transformed with the XbaI fragment from the plasmid pALK171.

The molecular weight of the phytase produced by Trichoderma differedfrom that produced by Aspergillus and the difference seemed to be due todifferences in the glycosylation level. The phytase secreted by T.reesei ALKO 233 was visible in the Western blots as three and thatsecreted by T. reesei ALKO 2221 as 6 to 9 major protein bands of sizesof about 45-65 kDal, the lowest of which corresponded in size thedeglycosylated Aspergillus phytase (45-48 kDal in SDS-PAGE). The phytasesecreted by T. reesei ATCC56765 was of a size of 65-80 kDal andconsisted of three to five protein bands. The molecular weight of thenative Aspergillus phytase run in SDS-PAGE is about 80-85 kDal. Thephytase protein produced by the transformants that had been transformedwith pALK172 fragment or pALK173A/B showed the same kind of bandingpattern.

III. Conclusions

The production level of Aspergillus phytase obtained when T. reesei wasused as a production host was surprisingly high. By using T. reesei ,the phytase is produced in a novel background differing from that ofAspergillus and containing enzymes important e.g. in feed applications.The molecular weight of the Aspergillus phytase protein produced inTrichoderma is different from that of Aspergillus. This difference insize seemed to be due to different glycosylation but did not affect theenzyme activity.

EXAMPLE 7 Production of Aspergillus niger pH 2.5 Acid Phosphatase inTrichoderma reesei

I. EXPERIMENTAL PROTOCOLS

1. Strains and plasmids

E. coli strains XL1 -Blue (Bullock, W. O. et al., Biotechniques 5:376-379 (1987); Stratagene, La Jolla, Calif., USA) and Sure™ (Greener,A., Strategies 3: 5-6 (1990); Stratagene, La Jolla, Calif., USA) wereused as hosts for constructions made of plasmids pALK601 (FIGS. 12 and13) and pAP-1 (FIGS. 12 and 13). The plasmid pALK601 contains the T.reesei cbh1 promoter, and terminator sequences and the Aspergillusnidulans acetamidase gene. The plasmid pAP-1 contains pH 2.5 acidphosphatase gene from Aspergillus niger var. awamori ALKO 243 (ATCC38854).

Trichoderma reesei strain ALKO 2221, a low aspartyl protease mutantderived from the T. reesei strain ALKO 233 (VTT-D-79125) byUV-mutagenesis was used as a recipient for the pH 2.5 acid phosphatasegene.

2. Growth media and culture conditions

E. coli strains were grown in L-broth (Maniatis, T., et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., USA (1982)) supplemented with ampicillin (50 μg/ml) whenneeded. E. coli cultivations were carried out at 37° C. overnight.

PD agar slants (Potato Dextrose broth by Difco, Detroit, Mich., USA)were used for storing the Trichoderma strains. The plates and media forT. reesei transformations were essentially as in Penttila et al.(Penttila, M., et al., Gene 61: 155-164 (1987)). The transformants werepurified on selective acetamide-CsCl medium (Penttila, M. et al., Gene61: 155-164 (1987) before transferring to PD slants. T. reeseitransformants were grown in lactose based medium (see Example 6,subparagraph 2) at 30° C. (250 rpm) for 7 days for expression of pH 2.5acid phosphatase under the control of the cbh1 promoter.

3. Manipulation of DNA

Manipulations of DNA were performed as described above for phytase,mainly by standard methods (Maniatis, T., et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., USA (1982)). Plasmid DNA from E. coli was isolated by using Qiagencolumns (Diagen GmbH, Dusseldorf, FRG) according to the supplier'sinstructions. For rapid screening of the plasmid DNA from E. coli, themethod of Holmes and Quigley, (Holmes and Quigley, Anal. Biochem. 114:193-197 (1981)) was used. The restriction enzymes, T4 DNA ligase, Klenowfragment of DNA polymerase I and T4 DNA polymerase used in the DNAmanipulations were from Boehringer (Mannheim, FRG) and New EnglandBiolabs (Beverly, Mass., USA). Each enzyme was used according to thesupplier's recommendation. DNA fragments for cloning or transformationswere isolated from low melting point agarose gels (FMC Bioproducts,Rockland, Me., USA) by the freeze thaw phenol method (Benson, S. A.Bio/Techniques 2: 66-68 (1984)) or by using the Mermaid™ Kit (BIO 101Inc., La Jolla, Calif., USA) according to the supplier's instructions.

Sequencing of the fusions between the cbh1 promoter and pH 2.5 acidphosphatase gene was carried out by means of pUC/M13 primers andextension primers using Taq DyeDeoxy™ Terminator Cycle Sequencing Kit(Applied Biosystems) and the automated sequencer (Applied Biosystems373A, Foster City, Calif., USA).

The oligonucleotides used were synthesized by an Applied Biosystems(Foster City, Calif., USA) 381A Synthesizer except the M13 primers thatwere purchased from the Applied Biosystems.

4. Transformations

Transformations were also performed as described above for phytase.Transformation of E. coli strains XL1-Blue or Sure was performed by thesupplier's method (Stratagene La Jolla, Calif., USA), T. reesei strainswere transformed essentially according to the method of Penttila et al.(Penttila, M., et al., Gene 61: 155-164 (1987)). Novozym 234 used infungal protoplast preparation for transformations was from Novo IndustriAS (Copenhagen, Denmark). Prior sporulating on PD slants T. reeseitransformants were purified through conidia on the selective acetamidemedium.

5. Enzyme activity assays

For the enzyme assays the mycelium was separated from the culture mediumby centrifuging for 15 min at 3,000 rpm (Sorvall SS-34, Dupont Company,Wilmington, Del., USA). The pH 2.5 acid phosphatase enzyme activity wasmeasured from the culture supernatant using paranitrophenylphosphate(Sigma, St. Louis, USA) as a substrate as described earlier. One pH 2.5acid phosphatase activity unit releases 1 nmol of inorganic phosphateper minute on the substrate p-nitrophenylphosphate in pH 2.5 at 37° C.One acid phosphatase normalized unit (APNU) is defined as the amount ofacid phosphatase activity produced by the A. niger ALKO 243 strain underthe cultivation conditions used (see Example 6, subparagraph 2).

Amyloglucosidase activity (AGU) was measured by using 1% Zulkowskystarch (Merck) as a substrate and measuring the amount of the releasedglucose units by boiling with DNS reagent (see below) after 10 min ofreaction at 60° C. at pH 4.8. Proteases (HUT) were measured at pH 4.7 asin Food Chemicals Codex (Food Chemicals Codex, National Academy Press,Washington, D.C., USA, pp. 496-497 (1981)) by using 2% haemoglobin(Sigma) as a substrate. Endoglucanase (ECU) and cellobiohydrolase (FPU)activities were measured as in IUPAC's Measurement of cellulaseactivities (IUPAC Commission on Biotechnology, Measurement of CellulaseActivities, Biochemical Engineering Research Centre, Indian Institute ofTechnology, Delhi, India, pp. 5-7 and 10-11 (1981)). 1%hydroxyethylcellulose (Fluka AG) in 50 mM Na-citrate buffer (pH 4) andWhatman no. 1 paper were used as substrates, respectively. DNS useddiffered from that described at the IUPAC's Measurement of cellulaseactivities (IUPAC Commission on Biotechnology, Measurement of CellulaseActivities, Biochemical Engineering Research Centre, Indian Institute ofTechnology, Delhi, India, pp. 5-7 and 10-11 (1984)) and was made byfirst diluting 50.0 g 2-hydroxy-3,5-dinitrobentsoicacid (Merck) into 4liters of deionized water. Then 80.0 g NaOH was added slowly by usingthe magnetic stirrer and 1,500 g K-Na-tartrate (Merck) was added anddiluted by heating the solution (maximum temperature 45° C.). The totalvolume was adjusted to 5 liters, the solution was filtered throughWhatman no. 1 and was protected from light.

6. SDS-page and Western blot analysis

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-page)and Western blot analysis were done according to the methods of Laemmli(Laemmli, U.K., Nature 227: 680-685 (1970)) and Towbin et al. (Towbin,H., et al., Proc. Natl. Acad. Sci. USA 76: 4350-4354 (1979)).Visualization of the pH 2.5 acid phosphatase protein in Western blotswas done by using the polyclonal rabbit antiserum KH1269. KH1269 wasmade against purified deglycosylated pH 2.5 acid phosphatase protein (M.Turunen, Alko Ltd.) and it was supplied by the National Public HealthInstitute (Helsinki, Finland). Visualization of the CBHI protein fromthe pH 2.5 acid phosphatase transformants in Western blots was done byusing the mouse monoclonal antibody CI-261 (Aho, S. et al., Eur. J.Biochem 200: 643-649 (1991)). Anti-rabbit-IgG and anti-mouse-IgGalkaline phosphate conjugate and color development substrates fromProtoBlot™ Immunoblotting system (Promega, Madison, USA) were used todetect the immunocomplexes.

7. PCR

The PCR reactions were performed by a Techne thermal cycler PHC-2(Techne Ltd., Cambridge, UK) in 100 μl volumes. The reaction mixturecontained 0.2 mM of each dNTP (Pharmacia pH 8.3), 20-50 pmol of eachprimer and 10 ng of plasmid template in 10 mM Tris buffer (pH 8.3), 50mM KCl, 1.5 mM MgCl₂ and 100 μg/ml gelatin. The protocol used was thefollowing: 96° C./10 min before adding the Taq DNA polymerase (2 units,Boehringer Mannheim, FRG) and 100 μl of paraffin oil, denaturation 95°C./1 min, annealing 60° C./1 min, extension 72° C./1 min for 30 cycles.The final extension time was 9 min to ensure completion of the strandsynthesis. The PCR fragments were purified by Mermaid™ Kit. The ends ofthe fragments were filled by using the DNA polymerase I Klenow fragment.

II. RESULTS

A. Vector constructions for overexpression of the pH 2.5 acidphosphatase gene in Trichoderma reesei ALKO 2221

1. Construction of plasmid pALK533

The plasmid pALK533 consists of Aspergillus niger var. awamori ALKO 243pH2.5 acid phosphatase gene with its own signal sequence inserted intothe Trichoderma reesei expression casette containing the cbh1 promoterand terminator sequences. pALK533 also contains the Aspergillus nidulansamdS gene as a selection marker for transformants.

The precise fusion between the cbh1 promoter and the pH 2.5 acidphosphatase signal sequence was done with PCR. The primers used for thePCR fragments are shown in FIG. 11. The SacII site in the cbh1 promoterarea was used in the 5'-primer and the MluI site of the acid phosphatasegene (374 nucleotides down from the N-terminal of the acid phosphatasegene) was used in the 3'-primer. The 5'-primer was a 39-mer containing atail of 19 nucleotides of the cbh1 promoter sequence preceding thesignal sequence joining exactly to the first 20 nucleotides of the acidphosphatase signal sequence. The 3'-primer was a 30-mer of pH 2.5 acidphosphatase gene. pAP-1 (FIG. 12) was used as a template in the PCRreactions. A fragment of the expected length of 466 bps was obtainedfrom the PCR reaction.

The 466 bp PCR fragment containing the pH 2.5 acid phosphatase signalsequence was digested with MluI and ligated to pAP-1 that had beendigested with HindIII, treated with DNA polymerase I Klenow fragment anddigested with MluI to obtain plasmid p102 (FIG. 12). The fusion and thePCR fragment were sequenced to ensure that no mistakes had occurred inthe PCR amplification.

To construct plasmid pALK533 (FIG. 12), a pH 2.5 acid phosphatase genecontaining the fusion was isolated from the plasmid p102 as an SphI(filled in with DNA polymerase I Klenow fragment)-SacII fragment andinserted between the cbh1 promoter and terminator of the plasmid pALK601(ΔNdeI) that had been digested with NdeI (filled in with DNA polymeraseI Klenow fragment) and SacII. In pALK601 (ΔNdeI), the NdeI site in theintron area of the amdS gene in pALK601 is inactivated using DNApolymerase I Klenow fragment. The linear fragment used fortransformations was digested out from the vector backbone with XbaI.

2. Construction of the plasmid pALK532

The plasmid pALK532 consists of the Aspergillus niger var. awamori ALKO243 pH 2.5 acid phosphatase gene inserted into the Trichoderma reeseiCBHI expression casette containing the cbh1 promoter and signal sequenceand terminator sequences. pALK532 also contains the Aspergillus nidulansamdS gene as a selection marker for transformants.

The precise fusion between the cbh1 signal sequence and the pH 2.5 acidphosphatase gene was done with PCR. The primers used for PCR fragmentsare shown in FIG. 11. The SfiI site in the cbh1 signal sequence was usedin the 5'-primer. The 5'-primer was a 46-mer containing a tail of 28nucleotides joining exactly to the first 18 nucleotides of the acidphosphatase N-terminal sequence. The 3'-primer was the same 30-mer usedin the construction of pALK533. pAP-1 was used as a template in the PCRreaction. A fragment of the expected length of 418 bps was obtained fromthe PCR reaction.

The 418 bp PCR fragment containing the cbh1 signal sequence was digestedwith MluI and ligated to pAP-1 that had been digested with HindIII,treated with DNA polymerase I Klenow fragment and digested with Mlul toobtain plasmid p51 (FIG. 13). The fusion and the PCR fragment weresequenced to ensure that no mistakes had occurred in the PCRamplification. To construct the plasmid pALK532 (FIG. 13), a pH 2.5 acidphosphatase fragment containing the fusion was isolated from the plasmidp51 as a SphI (filled in with DNA polymerase I Klenow fragment)-SfiIfragment and was inserted between the cbh1 promoter and terminator areasof the plasmid pALK601 (ΔNdeI). pALK601 (ΔNdeI) had been digested withNdeI and filled in with the DNA polymerase I Klenow fragment anddigested with SfiI. The approximately 7.8 kb linear fragment thatcontained no bacterial sequences was isolated from pALK532 byrestricting with XbaI and was used for transformations.

B. Transformation of Trichoderma reesei and screening of thetransformants

Trichoderma reesei ALKO 2221 was transformed separately with the linearXbaI fragments from the plasmid pALK532 and pALK533. Transformationfrequencies (transformants/μg of DNA) varied from 2 to 30.

Forty-four T. reesei ALKO 2221/pALK532 transformants and 103 pALK533transformants were purified through conidia and were cultivated in shakeflasks.

C. pH 2.5 acid phosphatase production by the Trichoderma transformants

The best transformants based on the pH 2.5 acid phosphatase productionare shown in the Table 10. The best enzyme activity level was 240APNU/ml in shake flask cultivation in lactose based medium.

                                      TABLE 10                                    __________________________________________________________________________    Strain   Plasmid                                                                            APNU/ml                                                                            CBHI(+/-)                                                                           AGU/ml                                                                             HUT/ml                                                                            ECU/ml                                                                            FPU/ml                                  __________________________________________________________________________    untransformed                                                                          none 0.2  (+)   48   18  600 3.8                                     ALKO 2221                                                                     transformed                                                                   SC-9     pALK532                                                                            240  (+)   53   32  380 2.4                                     KA-31    pALK533                                                                            240  (+)   37   19  490 2.0                                     KA-17    pALK533                                                                            230  (-)   44   52  760 1.1                                     KB-44    pALK533                                                                            230  (+)   37   16  490 ND                                      KB-18    pALK533                                                                            220  ND    ND   ND  ND  ND                                      SB-4     pALK532                                                                            210  (+)   35   14  590 ND                                      KA-28    pALK533                                                                            190  (+)   ND   ND  ND  ND                                      KB-38    pALK533                                                                            190  (+)   ND   ND  ND  ND                                      SC-6     pALK532                                                                            190  (+)   40   21  520 ND                                      SC-32    pALK532                                                                            190  (-)   ND   ND  ND  ND                                      A. niger ALKO 243                                                                      none 1    ND    46   31   35 0.0                                     __________________________________________________________________________     The best pH 2.5 acid phosphatase producing T. reesei ALKO 2221                transformants. pH 2.5 acid phosphatase enzyme activity levels as APNU/ml      produced in the 50 ml shake flask cultivations, background activities         (AGU, ECU, FPU and HUT/ml) and the production of CBHI protein (+/-,           Western blot) are shown.                                                 

Both the acid phosphatase and the cbh1 signal sequence worked equallywell and about the same level of pH 2.5 acid phosphatase activity couldbe achieved. The best pH 2.5 acid phosphatase activity level wasproduced by Trichoderma transformant SC-9 and was about 250 fold greaterthan the levels produced by native Aspergillus niger var. awamori ALKO243 strain in corresponding conditions.

Two out of the nine best producers did not react with the monoclonalCBHI antibody in Western blot analysis suggesting that the expressioncasette had integrated to the cbh1 locus in those two transformants(Table 10).

D. The enzyme background in the pH 2.5 acid phosphatase preparationsproduced by T. reesei

The pH 2.5 acid phosphatase is expressed in the T. reesei transformantsin high amounts and the background of some other enzyme activities inthe supernatants of T. reesei transformants is different from those inthe Aspergillus supernatant (Table 10). Both endoglucanase andcellobiohydrolase activities are significantly higher when T. reesei isused as a production host. The T. reesei transformants also producedproportionally less glucoamylase activity than the A. niger ALKO 243strain.

E. Identification of the pH 2.5 acid phosphatase produced by theTrichoderma transformants

Samples from the growth media of the transformants and the T. reeseiALKO 2221 strain were analyzed in Western blot (FIG. 14). The followingsamples were analyzed: 10 ng of purified Aspergillus ALKO 243 pH 2.5acid phosphatase; 10 ng of endoF treated Aspergillus ALKO 243 pH 2.5acid phosphatase; and 60 ng of protein from the each of the culturesupernatants of Trichoderma reesei ALKO 2221 transformants SC-9, KA-31,KA-17, KB-44, KB-18, SB-4 and KA-28 (FIG. 14).

The pH 2.5 acid phosphatase secreted by T. reesei transformants was seenas four protein bands of sizes of about 50-66 kD. This is probably dueto the different level of glycolysation of the protein part of thesecreted pH 2.5 acid phosphatase. Compared to the size of the pH 2.5acid phosphatase produced by Aspergillus niger var. awamori ALKO 243strain (66 kD) a majority of the pH 2.5 acid phosphatase proteinsproduced by T. reesei are smaller than that produced by Aspergillus.

All references are incorporated herein by reference. Having now fullydescribed the invention, it will be understood by those with skill inthe art that the scope may be performed with a wide and equivalent rangeof concentrations, parameters, and the like, without affecting thespirit or scope of the invention or any embodiment thereof.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 69                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2071 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: join(136..915, 970..1089, 1142..1245,                           1305..1737)                                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCATGCTGGACCGCAATCTCCGATCGCCGGGTATAAAAGGTCCTCCAAACCCCTCTCGGT60                CGATATGTACCCCGCTCGTCATCTCCAATCCTCTCGAGAGCACCTTCTCCAGCTTTTGTC120               AATTGTACCTTCGCAATGCCTCGCACCTCTCTCCTCACCCTGGCCTGTGCT171                        MetProArgThrSerLeuLeuThrLeuAlaCysAla                                          1510                                                                          CTGGCCACGGGCGCATCCGCTTTCTCCTACGGCGCTGCCATTCCTCAG219                           LeuAlaThrGlyAlaSerAlaPheSerTyrGlyAlaAlaIleProGln                              152025                                                                        TCAACCCAGGAGAAGCAGTTCTCTCAGGAGTTCCGCGATGGCTACAGC267                           SerThrGlnGluLysGlnPheSerGlnGluPheArgAspGlyTyrSer                              303540                                                                        ATCCTCAAGCACTACGGTGGTAACGGACCCTACTCCGAGCGTGTGTCC315                           IleLeuLysHisTyrGlyGlyAsnGlyProTyrSerGluArgValSer                              45505560                                                                      TACGGTATCGCTCGCGATCCCCCGACCAGCTGCGAGGTCGATCAGGTC363                           TyrGlyIleAlaArgAspProProThrSerCysGluValAspGlnVal                              657075                                                                        ATCATGGTCAAGCGTCACGGAGAGCGCTACCCGTCCCCTTCAGCCGGC411                           IleMetValLysArgHisGlyGluArgTyrProSerProSerAlaGly                              808590                                                                        AAGGACATCGAAGAGGCCCTGGCCAAGGTCTACAGCATCAACACTACT459                           LysAspIleGluGluAlaLeuAlaLysValTyrSerIleAsnThrThr                              95100105                                                                      GAATACAAGGGCGACCTGGCCTTCCTGAACGACTGGACCTACTACGTC507                           GluTyrLysGlyAspLeuAlaPheLeuAsnAspTrpThrTyrTyrVal                              110115120                                                                     CCTAATGAGTGCTACTACAACGCCGAGACCACCAGCGGCCCCTACGCC555                           ProAsnGluCysTyrTyrAsnAlaGluThrThrSerGlyProTyrAla                              125130135140                                                                  GGTTTGCTGGACGCGTACAACCATGGCAACGATTACAAGGCTCGCTAC603                           GlyLeuLeuAspAlaTyrAsnHisGlyAsnAspTyrLysAlaArgTyr                              145150155                                                                     GGCCACCTCTGGAACGGTGAGACGGTCGTGCCCTTCTTTTCTAGTGGC651                           GlyHisLeuTrpAsnGlyGluThrValValProPhePheSerSerGly                              160165170                                                                     TACGGACGTGTCATCGAGACGGCCCGCAAGTTCGGTGAGGGTTTCTTT699                           TyrGlyArgValIleGluThrAlaArgLysPheGlyGluGlyPhePhe                              175180185                                                                     GGCTACAACTACTCCACCAACGCTGCCCTCAACATCATCTCCGAGTCC747                           GlyTyrAsnTyrSerThrAsnAlaAlaLeuAsnIleIleSerGluSer                              190195200                                                                     GAGGTCATGGGCGCGGACAGCCTCACGCCCACCTGTGACACCGACAAC795                           GluValMetGlyAlaAspSerLeuThrProThrCysAspThrAspAsn                              205210215220                                                                  GACCAGACCACCTGCGACAACCTGACTTACCAGCTGCCCCAGTTCAAG843                           AspGlnThrThrCysAspAsnLeuThrTyrGlnLeuProGlnPheLys                              225230235                                                                     GTCGCTGCTGCCCGCCTAAACTCCCAGAACCCCGGCATGAACCTCACC891                           ValAlaAlaAlaArgLeuAsnSerGlnAsnProGlyMetAsnLeuThr                              240245250                                                                     GCATCTGATGTCTACAACCTGATGGGTATGTGATTACGGTACAATCATTGGCTC945                     AlaSerAspValTyrAsnLeuMet                                                      255260                                                                        AAACCTCCAGCTGACAGCATCCTAGTTATGGCCTCCTTTGAGCTCAATGCT996                        ValMetAlaSerPheGluLeuAsnAla                                                   265                                                                           CGTCCCTTCTCCAACTGGATCAACGCCTTTACCCAGGACGAATGGGTC1044                          ArgProPheSerAsnTrpIleAsnAlaPheThrGlnAspGluTrpVal                              270275280285                                                                  AGCTTCGGTTACGTTGAGGATTTGAACTACTACTACTGCGCTGGG1089                             SerPheGlyTyrValGluAspLeuAsnTyrTyrTyrCysAlaGly                                 290295300                                                                     TGAGTTTACCATTTGATCCATTATTGTCTTGGATCAGCTAACGATCGATAGTCCC1144                   Pro                                                                           GGTGACAAGAACATGGCTGCTGTGGGTGCCGTCTACGCCAACGCCAGT1192                          GlyAspLysAsnMetAlaAlaValGlyAlaValTyrAlaAsnAlaSer                              305310315                                                                     CTCACCCTCCTGAACCAGGGACCCAAGGAAGCCGGCTCCTTGTTCTTC1240                          LeuThrLeuLeuAsnGlnGlyProLysGluAlaGlySerLeuPhePhe                              320325330                                                                     AACTTGTACGTTCTCGGCAGAATCAGAGTCTCACAAAAAGAAACTCTTCACTAACA1296                  AsnPhe                                                                        335                                                                           TATAGTAGTGCCCACGACACCAACATCACCCCCATCCTCGCCGCCCTA1344                          AlaHisAspThrAsnIleThrProIleLeuAlaAlaLeu                                       340345                                                                        GGCGTCCTCATCCCCAACGAGGACCTTCCTCTTGACCGGGTCGCCTTC1392                          GlyValLeuIleProAsnGluAspLeuProLeuAspArgValAlaPhe                              350355360                                                                     GGCAACCCCTACTCGATCGGCAACATCGTGCCCATGGGTGGCCATCTG1440                          GlyAsnProTyrSerIleGlyAsnIleValProMetGlyGlyHisLeu                              365370375380                                                                  ACCATCGAGCGTCTCAGCTGCCAGGCCACCGCCCTCTCGGACGAGGGT1488                          ThrIleGluArgLeuSerCysGlnAlaThrAlaLeuSerAspGluGly                              385390395                                                                     ACCTACGTGCGTCTGGTGCTGAACGAGGCTGTACTCCCCTTCAACGAC1536                          ThrTyrValArgLeuValLeuAsnGluAlaValLeuProPheAsnAsp                              400405410                                                                     TGCACCTCCGGACCGGGCTACTCCTGCCCTCTGGCCAACTACACCTCC1584                          CysThrSerGlyProGlyTyrSerCysProLeuAlaAsnTyrThrSer                              415420425                                                                     ATCCTGAACAAGAATCTGCCAGACTACACGACCACCTGCAATGTCTCT1632                          IleLeuAsnLysAsnLeuProAspTyrThrThrThrCysAsnValSer                              430435440                                                                     GCGTCCTACCCGCAGTATCTGAGCTTCTGGTGGAACTACAACACCACG1680                          AlaSerTyrProGlnTyrLeuSerPheTrpTrpAsnTyrAsnThrThr                              445450455460                                                                  ACGGAGCTGAACTACCGCTCTAGCCCTATTGCCTGCCAGGAGGGTGAT1728                          ThrGluLeuAsnTyrArgSerSerProIleAlaCysGlnGluGlyAsp                              465470475                                                                     GCTATGGACTAGATGCAGAGGGGTAGGTCCCGGGATACTTTAGTGATGA1777                         AlaMetAsp                                                                     TTGATATTCAAGTTTGGTGGTGACGATCACCTTGTTAATAGTCTTGTACAGTCATACGGT1837              GAATGTAAATAATGATAATAGCAATGATACATGTTGGAATCTCGTTTTGTTCTTTGTGTG1897              CATAGGCGCTTTGGGGGTGTATTTTTAGGCGTTAGACTTATTTTCAATTCGTGTATAATG1957              CGGTCAGTAAATGAATCATCAATTATTCAAATGCAATGCTGTATACGTGAAACTATTGGG2017              TTAAGACGCAGCTACTAGCTGACTGCTTGGTTACTTTCTGTGTACACCGCATGC2071                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 479 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetProArgThrSerLeuLeuThrLeuAlaCysAlaLeuAlaThrGly                              151015                                                                        AlaSerAlaPheSerTyrGlyAlaAlaIleProGlnSerThrGlnGlu                              202530                                                                        LysGlnPheSerGlnGluPheArgAspGlyTyrSerIleLeuLysHis                              354045                                                                        TyrGlyGlyAsnGlyProTyrSerGluArgValSerTyrGlyIleAla                              505560                                                                        ArgAspProProThrSerCysGluValAspGlnValIleMetValLys                              65707580                                                                      ArgHisGlyGluArgTyrProSerProSerAlaGlyLysAspIleGlu                              859095                                                                        GluAlaLeuAlaLysValTyrSerIleAsnThrThrGluTyrLysGly                              100105110                                                                     AspLeuAlaPheLeuAsnAspTrpThrTyrTyrValProAsnGluCys                              115120125                                                                     TyrTyrAsnAlaGluThrThrSerGlyProTyrAlaGlyLeuLeuAsp                              130135140                                                                     AlaTyrAsnHisGlyAsnAspTyrLysAlaArgTyrGlyHisLeuTrp                              145150155160                                                                  AsnGlyGluThrValValProPhePheSerSerGlyTyrGlyArgVal                              165170175                                                                     IleGluThrAlaArgLysPheGlyGluGlyPhePheGlyTyrAsnTyr                              180185190                                                                     SerThrAsnAlaAlaLeuAsnIleIleSerGluSerGluValMetGly                              195200205                                                                     AlaAspSerLeuThrProThrCysAspThrAspAsnAspGlnThrThr                              210215220                                                                     CysAspAsnLeuThrTyrGlnLeuProGlnPheLysValAlaAlaAla                              225230235240                                                                  ArgLeuAsnSerGlnAsnProGlyMetAsnLeuThrAlaSerAspVal                              245250255                                                                     TyrAsnLeuMetValMetAlaSerPheGluLeuAsnAlaArgProPhe                              260265270                                                                     SerAsnTrpIleAsnAlaPheThrGlnAspGluTrpValSerPheGly                              275280285                                                                     TyrValGluAspLeuAsnTyrTyrTyrCysAlaGlyProGlyAspLys                              290295300                                                                     AsnMetAlaAlaValGlyAlaValTyrAlaAsnAlaSerLeuThrLeu                              305310315320                                                                  LeuAsnGlnGlyProLysGluAlaGlySerLeuPhePheAsnPheAla                              325330335                                                                     HisAspThrAsnIleThrProIleLeuAlaAlaLeuGlyValLeuIle                              340345350                                                                     ProAsnGluAspLeuProLeuAspArgValAlaPheGlyAsnProTyr                              355360365                                                                     SerIleGlyAsnIleValProMetGlyGlyHisLeuThrIleGluArg                              370375380                                                                     LeuSerCysGlnAlaThrAlaLeuSerAspGluGlyThrTyrValArg                              385390395400                                                                  LeuValLeuAsnGluAlaValLeuProPheAsnAspCysThrSerGly                              405410415                                                                     ProGlyTyrSerCysProLeuAlaAsnTyrThrSerIleLeuAsnLys                              420425430                                                                     AsnLeuProAspTyrThrThrThrCysAsnValSerAlaSerTyrPro                              435440445                                                                     GlnTyrLeuSerPheTrpTrpAsnTyrAsnThrThrThrGluLeuAsn                              450455460                                                                     TyrArgSerSerProIleAlaCysGlnGluGlyAspAlaMetAsp                                 465470475                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TAYTAYGGNCAYGGNGC17                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CARGGNGTNGGNTAYGC17                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TAYGTNGARATGATGCARAA20                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ATGATGCAAAATCAAGCTGAACA23                                                     (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2363 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: join(404..447, 550..1906)                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CATCCAGGCACCCTTTCCCAACGGGGGAACTTCCGTTGTCCACGTGCCCTGGTTCAGCCA60                ATCAAAGCGTCCCACGGCAATGCTGGATCAACGATCAACTTGAATGCAATAAATGAAGAT120               GCAACTAACACCATCTGTTGCCTTTCTCTCGAGAAAGCTCCTCCACTTCTCACACTAGAT180               TTATCCGTTCCTTGTCGACTTCCCGTCCCATTCGGCCTCGTCCACTGAAGATCTATCCCA240               CCATTGCACGTGGGCCACCTTTGTGAGCTTCTAACCTGAACTGGTAGAGTATCACACAAC300               ATGCGAAAGTGGGATGAAGGGGTTATATGAGGACCGTCCGGTCCGGCGCGATGGCCGTAG360               CTGCCAATCGCTGCTGTGCAAGAAATTTCTTCTCATAGGCATCATGGGCGTCTCT415                    MetGlyValSer                                                                  GCTGTTCTACTTCCTTTGTATCTCCTAGCTGGGTATGCTAAG457                                 AlaValLeuLeuProLeuTyrLeuLeuAlaGly                                             51015                                                                         CACCGCTATCTAAGTCTGATAAGGACCCTCTTTGCCGAGGGCCCCTGAAGCTCGGACTGT517               GTGGGACTACTGATCGCTGACAATCTGTGCAGAGTCACCTCCGGACTGGCA568                        ValThrSerGlyLeuAla                                                            20                                                                            GTCCCCGCCTCGAGAAATCAATCCACTTGCGATACGGTCGATCAAGGG616                           ValProAlaSerArgAsnGlnSerThrCysAspThrValAspGlnGly                              253035                                                                        TATCAATGCTTCTCCGAGACTTCGCATCTTTGGGGTCAATACGCGCCG664                           TyrGlnCysPheSerGluThrSerHisLeuTrpGlyGlnTyrAlaPro                              404550                                                                        TTCTTCTCTCTGGCAAACGAATCGGCCATCTCCCCTGATGTGCCCGCC712                           PhePheSerLeuAlaAsnGluSerAlaIleSerProAspValProAla                              556065                                                                        GGTTGCAGAGTCACTTTCGCTCAGGTCCTCTCCCGTCATGGAGCGCGG760                           GlyCysArgValThrPheAlaGlnValLeuSerArgHisGlyAlaArg                              70758085                                                                      TATCCGACCGAGTCCAAGGGCAAGAAATACTCCGCTCTCATTGAGGAG808                           TyrProThrGluSerLysGlyLysLysTyrSerAlaLeuIleGluGlu                              9095100                                                                       ATCCAGCAGAACGTGACCACCTTTGATGGAAAATATGCCTTCCTGAAG856                           IleGlnGlnAsnValThrThrPheAspGlyLysTyrAlaPheLeuLys                              105110115                                                                     ACATACAACTACAGCTTGGGTGCAGATGACCTGACTCCCTTCGGAGAG904                           ThrTyrAsnTyrSerLeuGlyAlaAspAspLeuThrProPheGlyGlu                              120125130                                                                     CAGGAGCTAGTCAACTCCGGCATCAAGTTCTACCAGCGATACGAATCG952                           GlnGluLeuValAsnSerGlyIleLysPheTyrGlnArgTyrGluSer                              135140145                                                                     CTCACAAGGAACATCATTCCGTTCATCCGATCCTCTGGCTCCAGCCGC1000                          LeuThrArgAsnIleIleProPheIleArgSerSerGlySerSerArg                              150155160165                                                                  GTGATCGCCTCCGGCGAGAAATTCATTGAGGGCTTCCAGAGCACCAAG1048                          ValIleAlaSerGlyGluLysPheIleGluGlyPheGlnSerThrLys                              170175180                                                                     CTGAAGGATCCTCGTGCCCAGCCGGGCCAATCGTCGCCCAAGATCGAC1096                          LeuLysAspProArgAlaGlnProGlyGlnSerSerProLysIleAsp                              185190195                                                                     GTGGTCATTTCCGAGGCCAGCTCATCCAACAACACTCTCGACCCAGGC1144                          ValValIleSerGluAlaSerSerSerAsnAsnThrLeuAspProGly                              200205210                                                                     ACCTGCACTGTCTTTGAAGACAGCGAATTGGCCGATACCGTCGAAGCC1192                          ThrCysThrValPheGluAspSerGluLeuAlaAspThrValGluAla                              215220225                                                                     AATTTCACCGCCACGTTCGCCCCCTCCATTCGTCAACGTCTGGAGAAC1240                          AsnPheThrAlaThrPheAlaProSerIleArgGlnArgLeuGluAsn                              230235240245                                                                  GACCTGTCTGGCGTGACTCTCACAGACACAGAAGTGACCTACCTCATG1288                          AspLeuSerGlyValThrLeuThrAspThrGluValThrTyrLeuMet                              250255260                                                                     GACATGTGCTCCTTCGACACCATCTCCACCAGCACCGTCGACACCAAG1336                          AspMetCysSerPheAspThrIleSerThrSerThrValAspThrLys                              265270275                                                                     CTGTCCCCCTTCTGTGACCTGTTCACCCATGACGAATGGATCCACTAC1384                          LeuSerProPheCysAspLeuPheThrHisAspGluTrpIleHisTyr                              280285290                                                                     GACTACCTCCAGTCCCTGAAAAAATACTACGGCCATGGCGCAGGTAAC1432                          AspTyrLeuGlnSerLeuLysLysTyrTyrGlyHisGlyAlaGlyAsn                              295300305                                                                     CCGCTCGGCCCGACCCAGGGCGTCGGCTACGCTAACGAGCTCATCGCC1480                          ProLeuGlyProThrGlnGlyValGlyTyrAlaAsnGluLeuIleAla                              310315320325                                                                  CGTCTCACCCACTCGCCTGTCCACGATGACACCAGCTCCAACCACACC1528                          ArgLeuThrHisSerProValHisAspAspThrSerSerAsnHisThr                              330335340                                                                     TTGGACTCGAACCCAGCTACCTTCCCGCTCAACTCTACTCTCTACGCG1576                          LeuAspSerAsnProAlaThrPheProLeuAsnSerThrLeuTyrAla                              345350355                                                                     GACTTTTCCCACGATAACGGCATCATCTCTATCCTCTTTGCTTTGGGT1624                          AspPheSerHisAspAsnGlyIleIleSerIleLeuPheAlaLeuGly                              360365370                                                                     CTGTACAACGGCACTAAGCCGCTGTCTACCACGACCGTGGAGAATATC1672                          LeuTyrAsnGlyThrLysProLeuSerThrThrThrValGluAsnIle                              375380385                                                                     ACCCAGACAGATGGGTTCTCGTCTGCTTGGACGGTTCCGTTTGCTTCG1720                          ThrGlnThrAspGlyPheSerSerAlaTrpThrValProPheAlaSer                              390395400405                                                                  CGTCTGTACGTCGAGATGATGCAGTGCCAGGCCGAGCAGGAGCCGCTG1768                          ArgLeuTyrValGluMetMetGlnCysGlnAlaGluGlnGluProLeu                              410415420                                                                     GTCCGTGTCTTGGTTAATGATCGCGTTGTCCCGCTGCATGGGTGTCCA1816                          ValArgValLeuValAsnAspArgValValProLeuHisGlyCysPro                              425430435                                                                     ATTGATGCTTTGGGGAGATGTACCCGGGATAGCTTTGTGAGGGGGTTG1864                          IleAspAlaLeuGlyArgCysThrArgAspSerPheValArgGlyLeu                              440445450                                                                     AGCTTTGCTAGATCTGGGGGTGATTGGGCGGAGTGTTCTGCT1906                                SerPheAlaArgSerGlyGlyAspTrpAlaGluCysSerAla                                    455460465                                                                     TAGCTGAACTACCTTGATGGATGGTATGTATCAATCAGAGTACATATCATTACTTCATGT1966              ATGTATTTACGAAGATGTACATATCGAAATATCGATGATGACTACTCCGGTAGATATTTG2026              GTCCCCTTCTATCCTTCGTTCCACAACCATCGCACTCGACGTACAGCATAATACAACTTC2086              AGCATTAACAAACGAACAAATAATATTATACACTCCTCCCCAATGCAATAACAACCGCAA2146              TTCATACCTCATATAGATACAATACAATACATCCATCCCTACCCTCAAGTCCACCCATCC2206              CATAATCAAATCCCTACTTACTCCTCCCCCTTCCCAGAACCCACCCCCGAAGGAGTAATA2266              GTAGTAGTAGAAGAAGCAGACGACCTCTCCACCAACCTCTTCGGCCTCTTATCCCCATAC2326              GCTATACACACACGAACACACCAAATAGTCAGCATGC2363                                     (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 467 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetGlyValSerAlaValLeuLeuProLeuTyrLeuLeuAlaGlyVal                              151015                                                                        ThrSerGlyLeuAlaValProAlaSerArgAsnGlnSerThrCysAsp                              202530                                                                        ThrValAspGlnGlyTyrGlnCysPheSerGluThrSerHisLeuTrp                              354045                                                                        GlyGlnTyrAlaProPhePheSerLeuAlaAsnGluSerAlaIleSer                              505560                                                                        ProAspValProAlaGlyCysArgValThrPheAlaGlnValLeuSer                              65707580                                                                      ArgHisGlyAlaArgTyrProThrGluSerLysGlyLysLysTyrSer                              859095                                                                        AlaLeuIleGluGluIleGlnGlnAsnValThrThrPheAspGlyLys                              100105110                                                                     TyrAlaPheLeuLysThrTyrAsnTyrSerLeuGlyAlaAspAspLeu                              115120125                                                                     ThrProPheGlyGluGlnGluLeuValAsnSerGlyIleLysPheTyr                              130135140                                                                     GlnArgTyrGluSerLeuThrArgAsnIleIleProPheIleArgSer                              145150155160                                                                  SerGlySerSerArgValIleAlaSerGlyGluLysPheIleGluGly                              165170175                                                                     PheGlnSerThrLysLeuLysAspProArgAlaGlnProGlyGlnSer                              180185190                                                                     SerProLysIleAspValValIleSerGluAlaSerSerSerAsnAsn                              195200205                                                                     ThrLeuAspProGlyThrCysThrValPheGluAspSerGluLeuAla                              210215220                                                                     AspThrValGluAlaAsnPheThrAlaThrPheAlaProSerIleArg                              225230235240                                                                  GlnArgLeuGluAsnAspLeuSerGlyValThrLeuThrAspThrGlu                              245250255                                                                     ValThrTyrLeuMetAspMetCysSerPheAspThrIleSerThrSer                              260265270                                                                     ThrValAspThrLysLeuSerProPheCysAspLeuPheThrHisAsp                              275280285                                                                     GluTrpIleHisTyrAspTyrLeuGlnSerLeuLysLysTyrTyrGly                              290295300                                                                     HisGlyAlaGlyAsnProLeuGlyProThrGlnGlyValGlyTyrAla                              305310315320                                                                  AsnGluLeuIleAlaArgLeuThrHisSerProValHisAspAspThr                              325330335                                                                     SerSerAsnHisThrLeuAspSerAsnProAlaThrPheProLeuAsn                              340345350                                                                     SerThrLeuTyrAlaAspPheSerHisAspAsnGlyIleIleSerIle                              355360365                                                                     LeuPheAlaLeuGlyLeuTyrAsnGlyThrLysProLeuSerThrThr                              370375380                                                                     ThrValGluAsnIleThrGlnThrAspGlyPheSerSerAlaTrpThr                              385390395400                                                                  ValProPheAlaSerArgLeuTyrValGluMetMetGlnCysGlnAla                              405410415                                                                     GluGlnGluProLeuValArgValLeuValAsnAspArgValValPro                              420425430                                                                     LeuHisGlyCysProIleAspAlaLeuGlyArgCysThrArgAspSer                              435440445                                                                     PheValArgGlyLeuSerPheAlaArgSerGlyGlyAspTrpAlaGlu                              450455460                                                                     CysSerAla                                                                     465                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CAACCGCGGACTGCGCATCATGGGCGTCTCTGCTGTTCT39                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ATTTCTCGAGGCGGGGACTGCC22                                                      (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CTCGGCCTTCTTGGCGAGAGCTCGTGCTCTGGCAGTCCCCGCCTCG46                              (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TTGGTGTCGACGGTGCTGGTGGAG24                                                    (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CTCGGCCTTCTTGGCCACAGCTCGTGCTTTCTCCTACGGCGCTGCC46                              (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GCCATGGTTGTACGCGTCCAGCAAACCGGC30                                              (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      CAACCGCGGACTGCGCATCATGCCTCGCACCTCTCTCCT39                                     (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GAATTCCCGGGACCTACCCCTCTGCAT27                                                 (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      LeuAlaValProAlaSerArgAsnGlnSerSerGlyAspThr                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ArgHisGlyXaaArgXaaPro                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      LysAspProArgAla                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      TyrTyrGlyHisLeuGlyAlaGlyAsnProLeuGlyProThrGln                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ThrGlyTyrValGlnAsnTyrValGlnMetGln                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6..7                                                            (D) OTHER INFORMATION: /label= Peptide                                        /note= "When deduced from the DNA sequence the                                amino acids at positions 6 and 7 were found to be                             serine."                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      AlaGlnProGlyGlnAlaAlaProLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      LeuTyrValGluMetMetGlnAsnGlnAlaGluGlnThrProLeuVal                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      LeuTyrValGluMetMetGlnCysGlnAlaGluGlnGluProLeuVal                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      PheIleGluGlyPheGlnSerAspLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      PheIleGluGlyPheGlnSerAspLys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      TyrAlaPheLeuLys                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      GlyLeuSerPheAlaArg                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      ValIleAlaSerGlyGluLys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      PheTyrGlnArg                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      PheTyrGlnArgAspSerPheValArg                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      AspSerPheValArg                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      ValLeuValAsnAsp                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      TyrGluSerLeuGln                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      TyrGluSerLeuThrArg                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      SerAlaAlaSerLeuAsnSer                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      LeuLysAspProArg                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      ValIleAlaSerGlyGluLys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      TyrProThrGluSerLys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      TyrPheAsnXaaGly                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 3..8                                                            (D) OTHER INFORMATION: /label= Peptide                                        /note= "The following are alternative amino acids                             at these positions: Proline at 3, Phenylalanine                               at 4, Serine at 6, Leucine at 7, and Valine at 8."                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      LeuGluAsnAspLeuAspGlyPheThrLeu                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      LeuGluAsnAspLeuSerGlyValThrLeuThr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 17                                                              (D) OTHER INFORMATION: /label= Peptide                                        /note= "The amino acid at position 17 may also be                             Tyrosine."                                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      TyrTyrGlyHisGlyAlaGlyAsnProLeuGlyProThrGlnGlyVal                              151015                                                                        GlyAlaAsnGlu                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      LeuIleAla                                                                     1                                                                             (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      ValThrPheAlaGlnValLeuSer                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      PheIleGluGlyPheGlnSerThr                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /label= Peptide                                        /note= "The amino acid at position 1 may also be                              Asparagine."                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      AspTyrLeuGlnSerLeuLys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      AsnIleGluProPheGlnValAsn                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      ValLeuValAsnAspArg                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      LeuAlaValProAlaSerArgAspGlnSerThrXaaAspThr                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /label= Peptide                                        /note= "When deduced from the DNA sequence the                                amino acid at position 1 was found to be                                      cysteine."                                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      ArgSerAla                                                                     1                                                                             (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      CARTTRCCNCARTTYAA17                                                           (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      PheSerTyrGlyAlaAlaIleProGlnSerThrGlnGluLys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      GlnPheSerGlnGluPheArgAspGlyTyr                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      TyrGlyGlyAsnGlyProTyr                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      ValSerTyrGlyIleAla                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      ArgHisGlyGluArgTyrProSerProSerAlaGlyLys                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      AspIleGluGluAlaLeuAlaLys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      AlaArgTyrGlyHisLeuTrpAsnGlyGluThr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      ValValProPhePheSerSerGly                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      PheSerSerGlyTyrGlyArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      GlnLeuProGlnPheLys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      ValAlaPheGlyAsnProTyr                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: modified.sub.-- base                                            (B) LOCATION: 12                                                              (D) OTHER INFORMATION: /mod.sub.-- base=i                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      GTRCCNCTYKCNATRGG17                                                           (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      CARCTNCCNCARTTYAA17                                                           (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      GAATTCCGAGTCCGAGGTCATGGGCGCG28                                                (2) INFORMATION FOR SEQ ID NO:67:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                      MetMetGlnCysGlnAlaGluGlnGluProLeuValArgValLeuVal                              151015                                                                        AsnAspArg                                                                     (2) INFORMATION FOR SEQ ID NO:68:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                      TyrAlaAsnGluLeuIleAla                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:69:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                      LeuAlaValProAlaSerArgAsnGlnSerThrCysAspThr                                    1510                                                                          __________________________________________________________________________

What is claimed is:
 1. A purified nucleic acid molecule comprising afirst genetic sequence encoding a phytase having the amino acid sequenceof amino acids 1-467 of SEQ ID NO: 8, or a catalytically active fragmentthereof.
 2. The purified nucleic acid molecule of claim 1, wherein saidamino acid sequence of amino acids 1-467 of SEQ ID NO: 8 is encoded bythe coding sequence of bases 404-1906 of SEQ ID NO:
 7. 3. The purifiednucleic acid molecule of claim 1, said catalytically active fragment ofsaid phytase having the amino acid sequence of amino acids 20-467 of SEQID NO:
 8. 4. The purified nucleic acid molecule of claim 3, wherein saidamino acid sequence of amino acids 20-467 is encoded by bases 563-1906of SEQ ID NO:
 7. 5. The purified nucleic acid molecule of claim 3,further comprising a second genetic sequence encoding a signal sequence,said second genetic sequence being operably linked to said first geneticsequence.
 6. The purified nucleic acid molecule of claim 5, wherein saidsecond genetic sequence encodes the phytase signal sequence having theamino acid sequence of amino acids 1-19 of SEQ ID NO:
 8. 7. The purifiednucleic acid molecule of claim 6, wherein the amino acid sequence ofamino acids 1-19 of SEQ ID NO: 8 is encoded by the coding sequence ofbases 404-562 of SEQ ID NO:
 7. 8. A vector comprising a first geneticsequence encoding a phytase having the amino acid sequence of aminoacids 1-467 of SEQ ID NO: 8, or a catalytically active fragment thereof.9. The vector of claim 8, wherein said amino acid sequence of aminoacids 1-467 of SEQ ID NO: 8 is encoded by the coding sequence of bases404-1906 of SEQ ID NO:
 7. 10. The vector of claim 8, said catalyticallyactive fragment of said phytase having the amino acid sequence of aminoacids 20-467 of SEQ ID NO:
 8. 11. The vector of claim 10, wherein saidamino acid sequence of amino acids 20-467 is encoded by bases 563-1906of SEQ ID NO:
 7. 12. The vector of claim 10, further comprising a secondgenetic sequence encoding a signal sequence, said second geneticsequence being operably linked to said first genetic sequence.
 13. Thevector of claim 12, wherein said second genetic sequence encodes thephytase signal sequence having the amino acid sequence of amino acids1-19 of SEQ ID NO:
 8. 14. The vector of claim 13, wherein the amino acidsequence of amino acids 1-19 of SEQ ID NO: 8 is encoded by the codingsequence of bases 404-562 of SEQ ID NO:
 7. 15. The vector of claim 8,wherein said vector is selected from the group consisting of pALK171,pALK172, pALK173A and pALK173B, and a fragment thereof that encodes saidphytase or said catalytically active fragment thereof.
 16. A purifiednucleic acid molecule comprising a first genetic sequence encoding a pH2.5 acid phosphatase having the amino acid sequence of amino acids 1-479of SEQ ID NO: 2, or a catalytically active fragment thereof.
 17. Thepurified nucleic acid molecule of claim 16, wherein said amino acidsequence of amino acids 1-479 of SEQ ID NO: 2 is encoded by bases136-1737 of SEQ ID NO:
 1. 18. The purified nucleic acid molecule ofclaim 16, said catalytically active fragment of said pH 2.5 acidphosphatase having the amino acid sequence of amino acids 20-479 of SEQID NO:
 2. 19. The purified nucleic acid molecule of claim 18, whereinsaid amino acid sequence of amino acids 20-479 is encoded by bases193-1737 of SEQ ID NO:
 1. 20. The purified nucleic acid molecule ofclaim 18, further comprising a second genetic sequence encoding a signalsequence, said second genetic sequence being operably linked to saidfirst genetic sequence.
 21. The purified nucleic acid molecule of claim20, wherein said second genetic sequence encodes the pH 2.5 acidphosphatase signal sequence having the amino acid sequence of aminoacids 1-19 of SEQ ID NO:
 2. 22. The purified nucleic acid molecule ofclaim 21, wherein the amino acid sequence of amino acids 1-19 of SEQ IDNO: 2 is encoded by the coding sequence of bases 136-192 of SEQ IDNO:
 1. 23. A vector comprising a first genetic sequence encoding a pH2.5 acid phosphatase having the amino acid sequence of amino acids 1-479of SEQ ID NO: 2, or a catalytically active fragment thereof.
 24. Thevector of claim 23, wherein said amino acid sequence of amino acids1-479 of SEQ ID NO: 2 is encoded by bases 136-1737 of SEQ ID NO:
 1. 25.The vector of claim 23, said catalytically active fragment of said pH2.5 acid phosphatase having the amino acid sequence of amino acids20-479 of SEQ ID NO:
 2. 26. The vector of claim 25, wherein said aminoacid sequence of amino acids 20-479 is encoded by bases 193-1737 of SEQID NO:
 1. 27. The vector of claim 25, further comprising a secondgenetic sequence encoding a signal sequence, said second geneticsequence being operably linked to said first genetic sequence.
 28. Thevector of claim 27, wherein said second genetic sequence encodes thephytase signal sequence having the amino acid sequence of amino acids1-19 of SEQ ID NO:
 2. 29. The vector of claim 28, wherein the amino acidsequence of amino acids 1-19 of SEQ ID NO: 2 is encoded by the codingsequence of bases 136-192 of SEQ ID NO:
 1. 30. The vector of claim 26,wherein said vector is selected from the group consisting of pALK532,pALK533, and a fragment thereof that encodes said pH 2.5 acidphosphatase or said catalytically active fragment thereof.
 31. A hostcell into which has been introduced a nucleic acid molecule comprising agenetic sequence encoding a phytate degrading enzyme selected from thegroup consisting of a phytase having the amino acid sequence of aminoacids 1-467 of SEQ ID NO: 8, a catalytically active fragment of saidphytase, a pH 2.5 acid phosphatase having the amino acid sequence ofamino acids 1-479 of SEQ ID NO: 2, and a catalytically active fragmentof said pH 2.5 acid phosphatase.
 32. The host cell of claim 31, whereinsaid genetic sequence has been introduced by transfection.
 33. The hostcell of claim 31, wherein said genetic sequence is introduced bytransformation.
 34. The host cell of claim 31, wherein said geneticsequence encodes a phytase having the amino acid sequence of amino acids1-467 of SEQ ID NO: 8, or a catalytically active fragment thereof. 35.The host cell of claim 34, wherein said amino acid sequence of aminoacids 1-467 of SEQ ID NO: 8 is encoded by the coding sequence of bases404-1906 of SEQ ID NO:
 7. 36. The host cell of claim 34, saidcatalytically active fragment of said phytase having the amino acidsequence of amino acids 20-467 of SEQ ID NO:
 8. 37. The host cell ofclaim 36, wherein said amino acid sequence of amino acids 20-467 isencoded by bases 563-1906 of SEQ ID NO:
 7. 38. The host cell of claim36, further comprising a second genetic sequence encoding a signalsequence, said second genetic sequence being operably linked to saidfirst genetic sequence.
 39. The host cell of claim 38, wherein saidsecond genetic sequence encodes the phytase signal sequence having theamino acid sequence of amino acids 1-19 of SEQ ID NO:
 8. 40. The hostcell of claim 39, wherein the amino acid sequence of amino acids 1-19 ofSEQ ID NO: 8 is encoded by the coding sequence of bases 404-562 of SEQID NO:
 7. 41. The host cell of claim 31, wherein said genetic sequenceencodes a pH 2.5 acid phosphatase having the amino acid sequence ofamino acids 1-479 of SEQ ID NO: 2, or a catalytically active fragmentthereof.
 42. The host cell of claim 41, wherein said amino acid sequenceof amino acids 1-479 of SEQ ID NO: 2 is encoded by bases 136-1737 of SEQID NO:
 1. 43. The host cell of claim 41, said catalytically activefragment of said pH 2.5 acid phosphatase having the amino acid sequenceof amino acids 20-479 of SEQ ID NO:
 2. 44. The host cell of claim 43,wherein said amino acid sequence of amino acids 20-479 is encoded bybases 193-1737 of SEQ ID NO:
 1. 45. The host cell of claim 41, furthercomprising a second genetic sequence encoding a signal sequence, saidsecond genetic sequence being operably linked to said first geneticsequence.
 46. The host cell of claim 45 wherein said second geneticsequence encodes the pH 2.5 acid phosphatase signal sequence having theamino acid sequence of amino acids 1-19 of SEQ ID NO:
 2. 47. The hostcell of claim 46 wherein the amino acid sequence of amino acids 1-19 ofSEQ ID NO: 2 is encoded by the coding sequence of bases 136-192 of SEQID NO:
 1. 48. The host cell of claim 31, wherein said host cell is aprokaryotic cell.
 49. The host cell of claim 31, wherein said host cellis a eukaryotic cell.
 50. The host cell of claim 49, wherein saideukaryotic cell is selected from the group consisting of an animal cell,a plant cell, a fungal cell, and a yeast cell.
 51. The host cell ofclaim 50, wherein eukaryotic cell is a fungal cell.
 52. The host cell ofclaim 51, wherein said fungal cell is Trichoderma.
 53. The host cell ofclaim 52, wherein said genetic sequence is integrated into the genome ofsaid Trichoderma.
 54. The host cell of claim 53, wherein said geneticsequence is integrated into the cbh1 locus of said Trichoderma.
 55. Thehost cell of claim 52, wherein said host cell is Trichoderma reesei. 56.The host cell of claim 52, wherein said first genetic sequence encodingsaid phytate degrading enzyme is operably linked to a second geneticsequence encoding a signal sequence.
 57. The host cell of claim 56,wherein said signal sequence is selected from the group consisting ofthe signal sequence of Trichoderma CBHI, the signal sequence ofTrichoderma CBHII, the signal sequence of Trichoderma EGI, the signalsequence of Trichoderma EGII, amino acids 1-19 of SEQ ID NO: 8, andamino acids 1-19 of SEQ ID NO:
 2. 58. A method for overexpressingphytate degrading enzymes in a host cell, wherein the method comprisesthe steps of:(a) preparing a DNA construct, said DNA constructcomprising a first genetic sequence selected from the group consistingof a phytase having the amino acid sequence of amino acids 1-467 of SEQID NO: 8, a catalytically active fragment of said phytase, a pH 2.5 acidphosphatase having the amino acid sequence of amino acids 1-479 of SEQID NO: 2, and a catalytically active fragment of said pH 2.5 acidphosphatase; (b) transforming a host cell with said DNA construct; and(c) cultivating the transformed host cell under conditions suitable forgrowth of said host cell and for expression of said phytate degradingenzyme.
 59. The method of claim 58, wherein said first genetic sequenceencodes a phytase having the amino acid sequence of amino acids 1-467 ofSEQ ID NO: 8, or a catalytically active fragment thereof.
 60. The methodof claim 59, wherein said amino acid sequence of amino acids 1-467 ofSEQ ID NO: 8 is encoded by the coding sequence of bases 404-1906 of SEQID NO:
 7. 61. The method of claim 59, said catalytically active fragmentof said phytase having the amino acid sequence of amino acids 20-467 ofSEQ ID NO:
 8. 62. The method of claim 61, wherein said amino acidsequence of amino acids 20-467 is encoded by bases 563-1906 of SEQ IDNO:
 7. 63. The method of claim 61, further comprising a second geneticsequence encoding a signal sequence, said second genetic sequence beingoperably linked to said first genetic sequence.
 64. The method of claim63, wherein said second genetic sequence encodes the phytase signalsequence having the amino acid sequence of amino acids 1-19 of SEQ IDNO:
 8. 65. The method of claim 64, wherein the amino acid sequence ofamino acids 1-19 of SEQ ID NO: 8 is encoded by the coding sequence ofbases 404-562 of SEQ ID NO:
 7. 66. The method of claim 58, wherein saidfirst genetic sequence encodes a pH 2.5 acid phosphatase having theamino acid sequence of amino acids 1-479 of SEQ ID NO: 2, or acatalytically active fragment thereof.
 67. The method of claim 66,wherein said amino acid sequence of amino acids 1-479 of SEQ ID NO: 2 isencoded by bases 136-1737 of SEQ ID NO:
 1. 68. The method of claim 66,said catalytically active fragment of said pH 2.5 acid phosphatase hangthe amino acid sequence of amino acids 20-479 of SEQ ID NO:
 2. 69. Themethod of claim 68, wherein said amino acid sequence of amino acids20-479 is encoded by bases 193-1737 of SEQ ID NO:
 1. 70. The method ofclaim 66, said DNA construct further comprising a second geneticsequence, said second genetic sequence being operably linked to saidfirst genetic sequence.
 71. The method of claim 70, wherein said secondgenetic sequence encodes the pH 2.5 acid phosphatase signal sequencehaving the amino acid sequence of amino acids 1-19 of SEQ ID NO:
 2. 72.The method of claim 71, wherein the amino acid sequence of amino acids1-19 of SEQ ID NO: 2 is encoded by the coding sequence of bases 136-192of SEQ ID NO:
 1. 73. The method of claim 58, wherein sad host cell is aprokaryotic cell.
 74. The method of claim 58, wherein said host cell isa eukaryotic cell.
 75. The method of claim 74, wherein said eukaryoticcell is selected from the group consisting of an animal cell, a plantcell, a fungal cell, and a yeast cell.
 76. The method of claim 75,wherein eukaryotic cell is a fungal cell.
 77. The method of claim 76,wherein said fungal cell is Trichoderma.
 78. The method of claim 77,wherein said DNA construct is provided to said Trichoderma in a linearform.
 79. The method of claim 78, wherein said linear form lacksbacterial sequences.
 80. The method of claim 77, wherein said DNAconstruct is provided to said Trichoderma in a circular plasmid form.81. The method of claim 77, wherein said host cell is Trichodermareesei.
 82. The method of claim 77, wherein said first genetic sequenceencoding said phytate degrading enzyme is operably linked to a secondgenetic sequence encoding a signal sequence.
 83. The method of claim 82,wherein said signal sequence is selected from the group consisting ofthe signal sequence of Trichoderma CBHI, the signal sequence ofTrichoderma CBHII, the signal sequence of Trichoderma EGI, the signalsequence of Trichoderma EGII, amino acids 1-19 of SEQ ID NO: 8, andamino acids 1-19 of SEQ ID NO:
 2. 84. The method of claim 77, whereinsaid DNA construct is provided by a vector selected from the groupconsisting of pALK171, pALK172, pALK173A, pALK173B, and a fragmentthereof that encodes the phytase having the amino acid sequence of aminoacids 1-467 of SEQ ID NO: 8, or a catalytically active fragment thereof.85. The method of claim 77, wherein said DNA construct is provided by avector selected from the group consisting of pALK532, pALK533, and afragment thereof that encodes the pH 2.5 acid phosphatase having theamino acid sequence of amino acids 1-479 of SEQ ID NO: 2, or acatalytically active fragment thereof.
 86. A method of cloning a fusionof the Trichoderma reesei cbh1 promoter to the Aspergillus niger var.awamori phytase signal sequence, wherein said method comprisespolymerase chain amplification of a fusion of the cbh1 promoter withphytase signal sequence using a 5' primer having SEQ ID NO: 9 and a 3'primer having SEQ ID NO:
 10. 87. A method of cloning a fusion of DNAencoding the Trichoderma reesei CBHI signal sequence to the Aspergillusniger var. awamori mature phytase coding sequence, wherein said methodcomprises polymerase chain amplification of a sequence encoding a fusionof the DNA encoding said CBHI signal sequence and said mature phytasecoding sequence with a 5' primer having SEQ ID NO: 11 and a 3' primerhaving SEQ ID NO:
 12. 88. A method of cloning a fusion of theTrichoderma reesei cbh1 promoter to the Aspergillus niger var. awamoripH 2.5 acid phosphatase signal sequence, wherein said method comprisespolymerase chain amplification of a sequence encoding a fusion of saidcbh1 promoter and said signal sequence with a 5' primer having SEQ IDNO: 15 and a 3' primer having SEQ ID NO:
 14. 89. A method of cloning afusion of DNA encoding the Trichoderma reesei CBHI signal sequence tothe Aspergillus niger var. awamori coding sequence of the mature pH 2.5acid phosphatase, wherein said method comprises polymerase chainamplification of a sequence encoding a fusion of the DNA encoding saidCBHI signal sequence and said mature pH 2.5 acid phosphatase codingsequence with a 5' primer having SEQ ID NO: 13 and a 3' primer havingSEQ ID NO:
 14. 90. A method of detecting a nucleic acid moleculeencoding a phytase, said method comprising(a) hybridizing DNA suspectedof encoding said phytase to DNA having the sequence of SEQ ID NO: 7; and(b) detecting the formation of the resulting double-stranded nucleicacid molecule.
 91. A method of detecting a nucleic acid moleculeencoding a pH 2.5 acid phosphatase, said method comprising(a)hybridizing DNA suspected of encoding said phytase to DNA having thesequence of SEQ ID NO: 1; and (b) detecting the formation of theresulting double-stranded nucleic acid molecule.